ES2967947T3 - Glycan polymers and related methods thereof - Google Patents

Abstract

Compositions of glycan polymers and methods for making and manufacturing them are described herein. Also provided are methods of treating a disease or disorder with a glycan polymer preparation. (Automatic translation with Google Translate, without legal value)

Description

DESCRIPTION

Glycan polymers and related methods thereof

Background

The microbiota of humans is complex and varies from person to person depending on genetics, age, sex, stress, nutrition and diet. The microbiota performs many activities and can influence host physiology. Modulation of the gut microbiota can alter community function and interaction with the host. A limited number of probiotic bacteria are known in the art and some association with health benefits when humans take probiotic bacteria is documented. Some foods are considered “prebiotic” foods and contain substances that can promote the growth of certain bacteria that are believed to be beneficial to the human host. The results of clinical trials with these substances are contradictory regarding their effectiveness and their influence on human health is generally described as modest.

Documents WO 2016/172657 and WO 2016/122889 refer to the use of glycan polymers to modulate the production of microbial metabolites. Document WO 2010/129839 refers to the use of alpha-(1,6) branched alpha-(1,2) oligodextran to improve the health of a subject or treat cholesterol-related diseases by increasing the level of chain fatty acids. short. EP 1597978 relates to the use of a glycan polymer preparation to increase levels of short chain fatty acids and treat various intestinal disorders.

Therefore, novel contributions are needed that can modulate the microbiota and improve human health.

Summary of the invention

The subject matter of the invention is as defined in the attached claims. Any matter outside the scope of the claims is provided for information purposes only.

Methods of treatment with a glycan polymer preparation or compositions thereof are used interchangeably with a glycan polymer preparation or compositions thereof for use in a treatment method.

Described herein are methods of treating a subject having a disease or disorder with a glycan polymer preparation, and compositions thereof.

In one aspect, the invention provides a glycan polymer preparation for use in a method of treating a subject having a disease or disorder associated with an unwanted level of a metabolite, wherein the metabolite is trimethylamine (TMA) or Trimethylamine N-oxide (TMAO), and wherein the disease or disorder is selected from the group consisting of atherosclerosis, cardiovascular disease, cardiovascular risk in HIV, carotid atherosclerosis, chronic heart disease, chronic heart failure, chronic kidney disease, disease chronic vascular disease, colorectal cancer, coronary heart disease, coronary artery disease (CAD), diabetes (type II), end-stage renal disease, HIV, inflammatory bowel disease, ischemic attack, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), obesity, radiation-induced acute intestinal symptoms (RIAIS) and stroke, said method comprising:

select a glycan polymer preparation based on or with the knowledge that it modulates the production or level of the metabolite, and

administering an amount of the glycan polymer preparation effective to result in a modulation of the level of the metabolite, thereby treating the disease or disorder,

wherein the glycan polymer preparation comprises glucose and at least one alpha-glycosidic bond, optionally, wherein the alpha-glycosidic bond is an alpha-1,3-glycosidic bond, alpha-1,4-glycosidic bond, or a combination thereof, and

where the average degree of polymerization (DP) of the preparation is between DP3-15.

In one embodiment, the glycan polymer is a substrate for a microbial constituent of the colon or intestine.

Brief description of the drawings

Figure 1 is a representative SEC curve between 16 and 20.5 minutes for a g lu l00 sample showing the average MW and the MW at 10% maximum absorption at both the leading and trailing edges of the curve. Figure 2 is a representative anomeric region of a 1H-13C HSQC spectrum of a glu100 sample showing the signal distribution of alpha and beta glycosidic bonds.

Figures 3A-3C are a representative anomeric region of a 1H-13C HSQC spectrum of glu50gal50 (Figure 3A), glu100 (Figure 3B) and gal 100 (Figure 3C) samples, demonstrating the additive effect of the peaks of fingerprints.

Figures 4A-4C are representative GC chromatograms of three representative permethylated and hydrolyzed glycans, glu50gal50 (Figure 4A), man52glu29gal19 (Figure 4B) and glu100 (Figure 4C), showing the distribution of regiochemistry as assigned by comparison with standards acquaintances.

Figure 5 is a graph showing a processed SEC profile comparing lactose (gray, beta-galacto-1,4-glucose) with a glycan prepared by treating lactose with beta-galactosidase as described in Example 2 (black).

Figure 6 is a graph showing a processed SEC profile comparing cellobiose (gray, beta-gluco-1,4-glucose) with a glycan prepared by treating cellobiose with beta-glucosidase as described in example 4 (black). The change in the maximum peak intensity of the DP2 materials is caused by the formation of allo-cellobiose (e.g., beta-gluco-1,6-glucose) which causes the average apparent Mw of the DP2 materials to change slightly.

Figures 7A-7B are graphs showing processed SEC profiles comparing (Figure 7A) maltobiose (gray, alpha-gluco-1,4-glucose) with a glycan prepared by treating maltobiose with alpha-glucosidase as described in example 5 (black), and (Figure 7B) maltobiose (gray) with a glycan from example 18 purified by yeast fermentation as described in example 9 (black). Although yeast can digest maltose, some DP2 materials remain due to trans-glycosylation in which maltose (alpha-gluco-1,4-glucose) is converted to allo-maltoses (e.g. alpha-gluco-1,6 -glucose; alpha-gluco-1,3-glucose) which is digested less efficiently by yeast.

Figure 8 is a graph showing a processed SEC profile comparing melibiose (gray, alpha-galacto-1,6-glucose) with a glycan prepared by treating melibiose with alpha-galactosidase as described in Example 3 (black). The change in the maximum peak intensity of the DP2 materials is caused by the formation of allo-melibiose (e.g. alpha-galacto-1,4-glucose) which causes the average apparent Mw of the DP2 materials to change slightly.

Figure 9 is an image showing fluorophore-assisted carbohydrate electrophoresis (FACE) analysis of a reverse hydrolysis reaction mixture of glucose by beta-glucosidase. Lane 1 is pure protein, and lanes 2-4 are reactions in trimethylphosphate, diethylene glycol dimethyl ether and tetraethylene glycol dimethyl ether respectively as described in example 7.

Figure 10 is a graph showing the SEC comparison of raw data of a glycan prepared by treating lactose with beta-galactosidase after 300 minutes with a glycan prepared by treating lactose with beta-galactosidase in the presence of d-galactose after 1200 minutes (i.e. at maximum conversion at DP>3) as described in example 8. The profile shows that the addition of D-galactose slows the reaction significantly but also shifts the product distribution towards increased amounts of DP>3 oligosaccharides.

Figure 11 is a graph showing processed SEC data related to the results of carbon fractionation of a glycan with the intention of removing the monomer from a sample without additional fractionation. The three curves represent the original glycan, the fraction of monomer removed from the original (m.p. of apparent peak -200) by 1% EtOH dissolution, and the remaining fraction by elution with 50% EtOH.

Figure 12 is a schematic representation of the synthesis of oligosaccharides by means of substrate-selective transglycosylation as described in Example 6. In each reaction, the enzymatic selectivity for transglycosylation of the non-reducing end monomer leads to different mixtures of products. . In this diagram, “A” and “B” could represent different monomers, different glycosidic bond stereochemistries, different glycosidic bond regiochemistries, or any combination thereof.

Figure 13 is a graph showing an SEC curve of a glycan prepared by treating lactose with betagalactosidase after 300 minutes as described in examples 11-18.

Figure 14 is a graph showing an SEC curve of a glycan prepared by treating lactose with beta-glucosidase after 300 minutes as described in examples 11-18.

Figure 15 is a graph showing the total genomes annotated and used in the Human Microbiome Project genomic analysis and the percentage of genomes by genus that encode each of the indicated metabolites.

Figures 16A-16B are graphs showing the percentage of genomes encoding CAZy families significantly enriched in butyrate producers (P < 0.001, Wilcox rank sum, FDR corrected and identified in > 10% of butyrate producers) . (Figure 16A) Percentage of butyrate producers and non-butyrate producers that encode at least 1 enzyme from the indicated family. (Figure 16B) Percentage of butyrate producers and non-butyrate producers encoding any CAZyme that is significantly enriched individually in butyrate producers.

Figure 17 is a graph showing the most abundant families in butyrate producers, ordered by average gene count. The graph represents the mean /- d. and.

Figures 18A-18B are graphs showing the percentage of genomes encoding CAZy families significantly depleted in TMA-lyase positive genomes (P < 0.05, Wilcox rank sum, FDR corrected). (Figure 18A) Percentage of TMA-lyase positive and negative genomes that encode at least 1 enzyme from the indicated family. (Figure 18B) Percentage of TMA-lyase positive and negative genomes encoding any CAZyme that is significantly depleted in TMA-lyase positive genomes.

Figure 19 is a graph showing the most abundant families in TMA-lyase negative genomes, ordered by average gene count. The graph represents the mean /- d. and.

Figures 20A-20B are graphs showing the percentage of genomes encoding CAZy families significantly depleted in urease-positive genomes (P < 0.05, Wilcox rank sum, FDR corrected). (Figure 20A) Percentage of urease-positive and -negative genomes encoding at least 1 enzyme from the indicated family. (Figure 20B) Percentage of urease positive and negative genomes encoding any CAZyme that is significantly depleted in urease positive genomes.

Figure 21 is a graph showing the most abundant families in urease-negative genomes, ordered by average gene count. The graph represents the mean /- d. and.

Figures 22A-22B are graphs showing the results of the LASSO linear regression model of SCFA production as a function of glycan composition, allowing for all 2nd order interaction terms. SCFA production in an ex vivo model (Figure 22A) and a defined community (Figure 22B). Figures 23A-23B are graphs showing the relative abundance of a Bacteroides cellulolyticus strain in a defined community composed of 15 strains, grown in the presence of carbohydrates for 48 hours (Figure 23A and Figure 23B, black circles), or in the presence of the indicated carbohydrates with an added glycan polymer preparation (e.g., Glu100) at 18 hours (Figure 23B, gray triangles). Mean ± dev relative abundance is shown. its T.

Figures 24A-24B are graphs showing the relative abundance of a Bacteroides cellulolyticus strain in a defined community composed of 14 strains. Figure 24A shows the relative abundance ofB. cellulolyticuscultured in the presence of various carbohydrates for 48 hours (black circles), or in the presence of the carbohydrates indicated withB. cellulolyticus added at 18 hours (gray triangles). Figure 24B shows the relative abundance ofB. cellulolyticuscultivated in the same defined community composed of 14 strains, in the presence of various carbohydrates andB. cellulolyticus added after 18 hours (black circles), or in the presence of the carbohydrates indicated withB. cellulolyticus added at 18 hours and glycan polymer preparation added (Glu, gray triangles). Mean ± dev relative abundance is shown. its T.

Figures 25A-25D are graphs showing the results of 16S rRNA sequencing analysis for the panel of bacteria examined in Example 23 and the correlation with butyrate production. As shown, several taxa are highly correlated with butyrate levels. (Figure 25A) Clostridiaceae (rho = 0.406 p value 0.003), (figure 25B) Lachnospiraceae roseburia (rho = 0.333 p value 0.018), (figure 25C) Bacteroides fragilis (rho = 0.483 p value 0), (figure 25D ) Turicibacteraceae turicibacter (rho = 0.554 p value = 0).

Figures 26A-26F are graphs showing the results of 16S rRNA sequencing analysis for the panel of bacteria examined in Example 23 and the correlation with acetate production. In the ex vivo assay, several taxa are highly correlated with acetate levels. As shown, several taxa are highly correlated with acetate levels. (Figure 26A) Clostridiaceae (rho = 0.428 p value = 0.002), (figure 26B) Bacteroides uniformis (rho = 0.525 p value = 0), (figure 26C) Ruminococcaceae OsciNospira (rho = -0.791 p value = 0) , (figure 26D)Bacteroides ovatus(rho = 0.405 p value 0.004), (figure 26E) Bacteroidales Rikenellaceae (rho = -0.739 p value = 0), (figure 26F) Clostridiales Ruminococcaceae (rho = -0.83 p value p = 0).

Figures 27A-27D are graphs showing the results of 16S rRNA sequencing analysis for the panel of bacteria examined in Example 23 and the correlation with propionate production. As shown, several taxa are highly correlated with propionate levels. (Figure 27A)Bacteroides ovatus(rho = 0.678 p value 0), (figure 27B)Bifidobacterium(rho = -0.781 p value =0), (figure 27C)Ruminococcus bromii(rho = -0.72 p value 0 ), (figure 27D) Bacteroides uniformis (rho = 0.559 p value = 0).

Figure 28. Number of CAZyme genes detected in spore-forming and non-spore-forming bacteria (mean) for each CAZyme family and subfamily. Only families are shown where genes were significantly enriched in spore-forming bacteria and were detected in >10% of spore-forming bacterial genomes (P < 0.05, Wilcox rank sum, FDR corrected).

Figures 29. Percentage of genomes encoding CAZy families significantly enriched in genomes of spore-forming bacteria versus non-spore-forming bacteria (P < 0.001, Wilcox rank sum, FDR corrected and identified in > 10% of spore-forming bacteria). spores). (A) Percentage of spore-forming and non-spore-forming bacteria that encode at least 1 enzyme from the indicated family. (B) Percentage of spore-forming and non-spore-forming bacteria encoding any CAZyme family or subfamily that are significantly enriched individually in spore-forming bacteria.

Figures 30A-30H are graphs showing the percentage of genomes encoding CAZy families significantly enriched in metabolite converting genomes (Figures 30A, 30C, 30E, 30G) and graphs showing the most abundant families in metabolite converting genomes, ordered by the average gene count (Figures 30B, 30D, 30F, 30H). The percentage of: converting and non-converting genomes of secondary bile acids is represented (figure 30<a>), genomes that encode CAZy families encoded exclusively in non-indole-producing bacteria (figure 30C), genomes that encode CAZy families significantly depleted in p-cresol-producing genomes (Figure 30E) and genomes encoding CAZy families significantly depleted in propionate-producing genomes (Figure 30G). Graphs are represented showing the most abundant families in: secondary bile acid converting genomes (Figure 30B), indole negative genomes (Figure 30D), p-cresol negative genomes (Figure 30F) and propionate negative genomes (Figure 30H). . The graph represents the mean /- d. and.

Figures 31A and 31B are graphs showing the relative abundance growth of Lachnospiraceae bacteria in an ex vivo community when grown in the presence of melibiose (e.g., melibiosa-1) (Figure 31A) or raffinose (e.g., raffinose-1). (Figure 31B) with alpha-galactooligosaccharides synthesized by alpha-galactosidase and either melibiose or raffinose. Figure 31A represents that enzymes 19 and 20 are alpha-galactosidases encoded in bacterial genomes of Lachnospiraceae and showed specific enrichment for those taxa (melibiosa-enz19-1 and melibiosa-enz20-1) compared to the alpha-galactosidases that were originated in other species (melibiosa-enz16-1 and melibiosa-enz17-1), which did not show the same specific enrichment for Lachnospiraceae bacteria. Figure 31B depicts that enzyme 19 is an alpha-galactosidase encoded in bacterial genomes of Lachnospiraceae and showed specific enrichment for those taxa (raffinose-enz19-1) compared to an alpha-galactosidase that originated from a different species (raffinose -enz16-1), which did not show the same specific enrichment for Lachnospiraceae bacteria.

Figures 32A-32D are graphs showing the relative abundance growth of Bifidobacterium (Figure 32A), Bacteroides (Figure 32B) and Roseburia (Figure 32C) bacteria in an ex vivo community when grown in the presence of lactulose (lactulose-1) and beta-galactooligosaccharides synthesized by betagalactosidase GH42 (enz23) and lactulose (lactulose-enz23-1). Enzyme 23 is a beta-galactosidase encoded in the bacterial genome of a species of Bifidobacteria and beta-galactooligosaccharides synthesized using this enzyme (lactulose-enz23-1) showed enrichment of Bifidobacterium, Roseburia and Bacteroides compared to lactulose alone. GH42 beta-galactooligosaccharides show enrichment in GH42 glycosidases from the genomes of Bacteroides and Firmicute (Figure 32D), common commensals of the gut microbiome.

Detailed description of the invention

The present invention presents, at least in part, methods of treating a subject having a disease or disorder (e.g., as described herein) with a glycan polymer preparation. In a first aspect, the invention provides a glycan polymer preparation for use in a method of treating a subject having a disease or disorder associated with an unwanted level of a metabolite, wherein the metabolite is trimethylamine (TMA). or trimethylamine N-oxide (TMAO), and wherein the disease or disorder is selected from the group consisting of atherosclerosis, cardiovascular disease, cardiovascular risk in HIV, carotid atherosclerosis, chronic heart disease, chronic heart failure, chronic kidney disease, chronic vascular disease, colorectal cancer, coronary heart disease, coronary artery disease (CAD), diabetes (type II), end-stage renal disease, HIV, inflammatory bowel disease, ischemic attack, metabolic syndrome, nonalcoholic fatty liver disease (NAFLD) , obesity, radiation-induced acute intestinal symptoms (RIAIS) and stroke,

understanding said method:

select a glycan polymer preparation based on or with the knowledge that it modulates the production or level of the metabolite, and

administering an amount of the glycan polymer preparation effective to result in a modulation of the level of the metabolite, thereby treating the disease or disorder,

wherein the glycan polymer preparation comprises glucose and at least one alpha-glycosidic bond, optionally, wherein the alpha-glycosidic bond is alpha-1,3-glycosidic bond, alpha-1,4-glycosidic bond, or a combination of the same, and

where the average degree of polymerization (DP) of the preparation is between DP3-15.

In some embodiments, the glycan polymer preparation is selected on the basis that it modulates an undesired level of a metabolite, where the metabolite is trimethylamine (TMA) or trimethylamine N-oxide (TMAO). The unwanted level of metabolite may be too high or too low. In some embodiments, the metabolite is associated with a desired (e.g., beneficial) effect on the health of the subject. In other embodiments, the metabolite is associated with an unwanted (e.g., harmful) effect on the health of the subject. In some embodiments, the methods described herein include increasing a metabolite. In other embodiments, the methods include decreasing a metabolite. In some embodiments, the metabolite is a microbial (e.g., bacterial) metabolite. In some embodiments, a first metabolite (e.g., produced by taxa A) is modulated to modulate a second metabolite (e.g., produced by taxa B). In some embodiments, the second metabolite is associated with a disease or disorder. The unwanted level of metabolite may occur anywhere in a subject's body (for example, the GI tract, including the colon and intestines, fecal matter, blood, brain, nervous system, an organ, including the heart, liver and kidneys, urine, and other part). In some embodiments, metabolite production by the microbiota is modulated (e.g., in the intestine) and has a local effect on metabolite levels (e.g., a local decrease or increase in the metabolite). In some embodiments, metabolite production by the microbiota is modulated (e.g., in the gut) and has a systemic effect on metabolite levels (e.g., a systemic decrease or increase in the metabolite). In some embodiments, modulation of a first metabolite (e.g., metabolite A, e.g., in the intestine) leads to a modulation of a second metabolite (e.g., metabolite B, e.g., in a body site other than the intestine). ). In some embodiments, preparations of glycan polymers are administered to subjects in need, wherein the glycan polymers are substrates (e.g., preferred substrates) for a glycosidase machinery specific to a class of microbial metabolite producers. In some embodiments, preparations of glycan polymers are administered to subjects in need, wherein the glycan polymers are substrates (e.g., preferred substrates) for a glycosidase machinery specific to a class of non-producers of a microbial metabolite. In some embodiments, the balance (e.g., the relative abundance of microbial taxa at a body site, such as, for example, the intestine) of metabolite producers relative to non-metabolite producers is modulated to modulate metabolite levels. produced by the site. In some embodiments, modulating the balance of producers relative to non-producers to modulate metabolite levels treat a disease or disorder that is associated with a dysregulation of the metabolite. In some embodiments, the subject has a dysbiosis of the site, such as the intestine. Further provided herein are preparations of glycan polymers that are substrates (e.g., preferred substrates) of producers or non-producers of microbial metabolites. In some embodiments, the glycan polymer preparations are tailored to the glycosidase enzyme profile of microbial or metabolite-producing or non-producing taxa, respectively, that is, the glycan polymers are substrates (e.g., preferred substrates) of the glycosidases. present in the genome of the producer or non-producer. In some embodiments, the glycosidases are enriched in or unique to one class (e.g., metabolite producer) relative to the other class (e.g., non-producer). Further provided herein are coformulations (e.g., symbiotic) of tailored glycan polymers and microbial taxa with a repertoire of glycosidases (glycosidase profile) capable of (preferentially) using the glycan polymers as a substrate. In some embodiments, the coformulations are used to increase the engraftment of microbial taxa into a microbial site, such as, for example, the intestine.

The glycan polymers described herein can be tailored to target a particular gut microbe, for example, a human gut microbe. In some embodiments, glycoside hydrolase (glycosidase) enzymes are selected to tailor a glycan polymer to a particular microbe. In some embodiments, the glycoside hydrolase (glycosidase) profile of a microbe is determined and a glycan polymer is tailored thereto, for example, using (e.g., in vitro) one or more glycoside hydrolase (glycosidase) so identified to produce a glycan polymer preparation under conditions that are suitable for producing glycan polymers. Glycoside hydrolases can be isolated (and optionally immobilized, for example, on a suitable substrate). In some embodiments, glycoside hydrolases can be extracted from a microbe (for example, a microbial extract comprising glycoside hydrolases). In some embodiments, microbial cells (e.g., bacteria) comprising glycoside hydrolases on their surface and/or intracellularly) may be used. In some embodiments, supernatants comprising glycoside hydrolases (e.g., from microbial cultures) may be used. In some embodiments, the glycoside hydrolase (glycosidase) profile of a particular microbe is not known or has not been determined, but enzymes derived from the microbe are used (e.g., in isolated, extracted, whole cell, supernatant, etc. forms). .) to produce the glycan polymers in the methods described herein. In some embodiments, the glycan polymer preparations produced as described herein are specific substrates for a particular microbe (or a group of microbes, e.g., a group of microbes with a comparable or similar glycosidase profile) and its glycosidase machinery. In some embodiments, the glycan polymer preparations are fermented specifically by the microbe or group of microbes, for example, in the GI tract of a human subject (for example, the glycan polymers are fermented at a faster rate or at a higher rate). degree compared to another microbe (or group of microbes), for example, with a different glycosidase profile). In some embodiments, glycan polymer preparations confer a growth advantage to the particular microbe. In some embodiments, glycan polymers can be used to modulate the production of a microbial metabolite, for example, a metabolite produced by the particular microbe, or a microbial metabolite not produced by the particular microbe. In the latter case, the particular microbe may compete with another microbe, one that produces an unwanted microbial metabolite, and successful competition for the particular microbe may lead to lower levels of the microbial metabolite. In some embodiments, glycan polymers can be used to promote engraftment into a subject's microbiota (e.g., gut microbiota, e.g., colonic microbiota) of a particular microbe that is administered to a subject in need of engraftment. In some embodiments, the glycan polymers confer a growth advantage to the particular microbe that allows it to compete successfully for, for example, space and nutrients, to more successfully engraft into the existing microbiota of the graft site (for example, the intestine).

Definitions

The present invention will be described with respect to particular embodiments and with reference to certain figures, but the invention is not limited thereto but only by the claims. The terms set forth below in this document should generally be understood in their common sense unless otherwise indicated.

“Abundance” of a microbial taxon as used herein is a relative term and refers to the relative presence of a microbial taxon with respect to other taxa in a community in a defined microbial niche, such as the GI tract, or in the entire host organism (for example, a human or laboratory animal disease model).

“Acquire” or “acquisition,” as the terms are used herein, refers to obtaining possession of a security, for example, a numerical value, an image, or a physical entity (for example, a sample), “ directly acquiring” or “indirectly acquiring” the security or physical entity. “Acquire directly” means performing a process (for example, performing a synthetic or analytical method or protocol) to obtain the security or physical entity. “Indirectly acquire” means receiving the security or physical entity from another party or source (for example, a third-party laboratory that directly acquired the physical entity or security). Directly acquiring a security or physical entity includes performing a process that includes a physical change to a physical substance or the use of a machine or device. Examples of direct acquisition of a value include obtaining a sample from a human subject. Directly acquiring a value includes performing a process that uses a machine or device, for example, an NMR spectrometer to obtain an NMR spectrum.

“Distinct,” as used herein, for example, with reference to one species in a glycan polymer, is intended to indicate that it is chemically and/or structurally different from another. For example, two sugars are “distinct” if they are chemically different, e.g., a fucose and a xylose, or structurally different, e.g., cyclic vs. acyclic, L-form vs. D. Two dimers are distinct if they consist of the same two monomers but one pair contains an alpha-1,4 bond and the other contains a beta-1,6 bond. The distinct entities may have any other suitable distinguishing characteristic or property that can be detected by methods known in the art and/or described herein.

As used herein, a “dosing regimen,” “dose regimen,” or “treatment regimen” is a drug delivery modality that achieves a therapeutic goal. A dosage regimen includes the definition of one, two, three or four of: a route of administration, a unit dose, a dosing frequency and a duration of treatment.

“Dysbiosis” refers to an unbalanced state of the microbiota, for example, within the GI tract, in which normal diversity, the proportion of a first bacterial taxon to a second bacterial taxon and/or function (e.g. , the production of a metabolite) of the ecological network is altered or disturbed. This unwanted state, e.g., unhealthy, may be due to several factors including, but not limited to, a decrease or increase in the diversity of the microbiota (e.g., bacterial taxa), overgrowth of one or more pathogens or pathobionts or the change to an ecological microbial community that no longer provides an essential function to the host subject and, in one embodiment, therefore no longer promotes health or that is associated with unwanted symptoms in the subject. In one embodiment, the production of a metabolite is modulated to contribute to the development of a disease or disorder.

By the terms "effective amount" and "therapeutically effective amount" of a composition (such as, for example, a pharmaceutical composition) or a pharmacological agent, is meant a sufficient amount of the composition or agent to provide the desired effect. In some embodiments, a physician or other healthcare professional decides the appropriate amount and dosage regimen. An effective amount also refers to an amount of a composition (such as, for example, a pharmaceutical composition) or a pharmacological agent that prevents the development or relapse of a medical condition.

“Microbial grafting” or simply “grafting” refers to the establishment (e.g., growth) of microbial taxa in a target niche (e.g., the human intestine, such as the colon or intestines) that are either underrepresented (e.g. , relative to a healthy reference subject) or absent (e.g., undetectable) in a human subject prior to engraftment (e.g., by administering the microbial taxa to the subject, e.g., in the form of a synbiotic described herein ). The grafted microbial taxa may become established during a transient period or demonstrate long-term stability in the microbiota populating the subject after grafting of the microbial taxa. In some embodiments, the grafted microbial taxa can induce an environmental change in the target niche that represents a change from a dysbiosis to a healthy state.

"Fructooligosaccharide" or "FOS", as the terms are used herein, refers to a fructose polymer, optionally comprising terminal glucose, of the following sequence: (Fru)n-Glc consisting of one or more of: beta 2,1, beta 2,6, alpha 1,2 and beta-1,2 glycosidic bonds, where n is usually 3-10. Variants include p-1,2 inulin-type and p-2,6 levan-type linkages between fructosyl units in the main chain. In one embodiment, the FOS is prepared by a method described in any of references 8,24,25, 61,67,69, 72,170 or 176-186, or 21,29, 170, 176 or 222 of Meyer, Biotechnological Production of Oligosaccharides - Applications in the Food Industry, chapter two, Food technology and Industry, 2015, (Meyer 2015). In one embodiment, the FOS is a FOS described in Sangeethaet al., or prepared by a method described in Sangeethaet al., 2005, 2014 found in Diez-Municioet al., 2014, Synthesis of novel bioactive lactose-derived oligosaccharides by microbial glycoside hydrolases , 2014, Microbial Biotechnology, 7:315-313 (Diez-Municioet al.2014). In one embodiment, FOS is prepared from a B enzyme. macerans, Z. mobilis, L. reutri, A. niger, A. japónicas, A. foetidus, A. sydowi, b A. Pullans, C. purpurea, F. oxysporum P. citrinum, P. frequentens, P. spinulosum, P. rigulosum, P. parasitica, S. brevicaulis, S. cerevisiaeoK. marxianus. In some embodiments, FOS is produced through the enzymatic action of a fructosyltransferase, pfructofuranosidase (EC 3.2.1.26), inulosuscrase (EC 2.4.1.9), levansucrase (EC 2.4.1.10) or endoinulinase.

“Galactooligosaccharide” or “GOS”, as the terms are used herein, refers to a mixture of substances produced from lactose, with two to eight saccharide units, in which one of the units is a terminal glucose and the remaining units are galactose and disaccharides comprising two galactose units. In one embodiment, GOS is a mixture of galactopyranosyl oligomers (DP = 3-8) linked primarily by p-(1,4) or p-(1,6) linkages, although low proportions of p(1,6) linkages may also be present. 1,2) or p-(1,3). The terminal glucosyl residues are linked by P-(1,4) bonds to galactosyl units. GOS is synthesized by the reverse action of p-galactosidases (EC 3.2.1.23) on lactose at relatively high lactose concentrations. In one embodiment, GOS is synthesized by the enzymatic action of a p-galactosidase from Bifidobacterium, e.g., Bifidobacterium longum, Kluyveromyces sp., Kluyveromyces marxianus, Aspergillus sp., e.g., Aspergillus oryzae, Escherichia coli K-12, Bacillus circulans, Lactobacillus bulgaricus, S. singularis, S. thermophilesoC. laurentii. In one embodiment, GOS is a GOS disclosed or prepared by a method described in any of references 8,105, 196-206 or 105,120, 198, 202-205 or 223-227 of Meyer 2015. S is a GOS described in or prepared by a method described in Panesaret al.2006 or Torreset al2010, 2014 found in Diez-Municioet al.2014.

“Glucooligosaccharide” or “GLOS,” as the terms are used herein, refers to a polymer of glucose subunits. The main unions in GLOS are (Glc)n [a(1^2), a(1^3), a(1^4) and a(1^6)]. In one embodiment, GLOS is prepared with dextransucrase (EC 2.4.1.5). In one embodiment, enzymes from bacteria (L. mesenteroides; L. citreum) can be used to produce GLOS. In one embodiment, GLOS is a GLOS described in, or prepared by a method described in, any of the references Remaudet al.,1992, Chung and Day, 2002 or Kimet al.,2014 found in Diez-Municioet al.,2014, Synthesis of novel bioactive lactose-derived oligosaccharides by microbial glycoside hydrolases, 2014, Microbial Biotechnology, 7:315-313.

As used herein, a “glycan polymer preparation” (also called “glycan polymer preparation”, “glycan preparation” or “glycan polymer”) is a preparation comprising glycan polymers having a desired effect (for example, a therapeutic effect). In some embodiments, the glycan polymer preparations do not contain one or more naturally occurring oligosaccharides, including: glucooligosaccharide, mannanoligosaccharide, inulin, lichnose, maltotretraose, nigerotetraose, nystose, sesemose, stachyose, isomaltotriose, nigerotriose, maltotriose, melezitose, maltotriulose, raffinose, kestose, fructooligosaccharide, 2'-fucosyllactose, galactooligosaccharide, glycosyl, idraparinux, isomaltooligosaccharide, maltodextrin, xylooligosaccharide, agar, agarose, alginic acid, alguronic acid, alpha-glycan, amylopectin, amylose, arabioxylan, beta-glucan, callose , capsulan, carrageenan, cellodextrin, cellulin, cellulose, chitin, chitin nanofibrils, chitin-glucan complex, chitosan, chrysolaminarin, curdlan, cyclodextrin, alpha-cyclodextrin, dextran, dextrin, dialdehyde starch, ficoll, fructan, fucoidan, galactoglucomannan, galactomannan, galactosaminogalactan, gellan gum, glucan, glucomannan, glucoronoxyland, glycocalyx, glycogen, hemicellulose, hypromellose, icodextrin, kefiran, laminarin, lentinan, levan polysaccharide, lichenin, mannan, mucilage, natural gum, paramylon, pectic acid, pectin, pentalstarch , phytoglycogen, pleuran, polygeenan, polydextrose, porphyran, pullulan, schizophyllan, sepharose, sinistrin, sizophyran, sugammadex, welan gum, xantham gum, xylan, xyloglucan, zymosan and the like. In some embodiments, a glycan polymer exists as a salt, for example, a pharmaceutically acceptable salt.

A “glycan subunit,” as used herein, refers to the individual unit of a glycan species disclosed herein, for example, the building blocks from which the glycan species. In one embodiment, a glycan subunit is a monomer. In one embodiment, a glycan subunit is a dimer. In one embodiment, a glycan subunit is a monosaccharide. In one embodiment, a glycan subunit is a disaccharide. In some embodiments, the glycan subunit is a carbohydrate and may be selected from a sugar alcohol, a short chain fatty acid, a sugar acid, an iminosugar, a deoxysugar, and an aminosugar. In some embodiments, the glycan subunit is erythrose, threose, erythulose, arabinose, lyxose, ribose, xylose, ribulose, xylulose, allose, altrose, galactose, glucose, gulose, idose, mannose, thallose, fructose, psychose, sorbose, tagatose. , fucose, fuculose, rhamnose, mannoheptulose, sedoheptulose and the like. In some embodiments, the glycan subunit is glucose, galactose, arabinose, mannose, fructose, xylose, fucose, or rhamnose. In some embodiments, a glycan comprises distinct glycan subunits, for example, a first and a second monosaccharide, or a first and a second disaccharide. In some embodiments, a glycan comprises distinct glycan subunits, for example, a first, second, third, fourth, and/or fifth distinct glycan subunit.

As used herein, “a glycosidase enzyme molecule” comprises a polypeptide that retains or has a glycosidase enzyme activity, for example, retains or has at least about 40%, about 50%, about 60%, approximately 70%, approximately 80%, approximately 90%, approximately 95%, approximately 99%, approximately 99.9% of the glycosidase enzyme turnover rate, or retains or has at least about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 99.9% of the specificity of the glycosidase enzyme, or retains or has at least about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, approximately 99.9% of the affinity for a glycan subunit of the glycosidase enzyme. In some embodiments, a glycosidase enzyme molecule comprises a polypeptide having a glycosidase enzyme activity that is at least about 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%. 190%, 200%, 300%, 400%, or 500% of the glycosidase enzyme turnover rate or has at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180 %, 190%, 200%, 300%, 400% or 500% of the affinity for a glycan subunit of the glycosidase enzyme. In some embodiments, the glycosidase enzyme molecule is a fragment (e.g., an active fragment) of the glycosidase enzyme. In some embodiments, the glycosidase enzyme molecule differs by at least 1, 2, 3, 4, 5, 10, 25, 50, 75, 100 or more amino acid residues compared to the glycosidase enzyme. In some embodiments, the glycosidase enzyme molecule comprises at least 1, 2, 3, 4, 5, 10, 25, 50, 75, 100 amino acid mutations (e.g., deletions, additions or substitutions) compared to the glycosidase enzyme. .

“Glycosidase enzymes” as used herein include glycosidases (also called “glycoside hydrolase” (GH)), glycosyltransferases (GT), and lysases.

As used herein, “glycotaxa” refers to bacterial microbes (e.g., human gut microbes) grouped according to the presence or absence (e.g., lack of) of a metabolic (e.g., enzymatic) function. In some embodiments, taxa may be grouped based on CAZy glycosidase/glycohydrolase (GH) or CAZy glycosyltransferase (GT) enzyme function. In some embodiments, the bacterial taxa may belong to any of class 1 glycotaxons, class 2 glycotaxons, class 3 glycotaxons, class 4 glycotaxons, class 5 glycotaxons, class 6 glycotaxons, or class 7 glycotaxons. In some embodiments, the class 1 glycotaxons contain the bacterial taxa that contain the buty/obuk genes. In some embodiments, class 2 glycotaxons contain gencutC-negative bacterial taxa. In some embodiments, class 3 glycotaxons contain genurease-negative bacterial taxa. In some embodiments, class 4 glycotaxons exclude one or more enzymes associated with propionate production selected from propionate kinase, propionate CoA-transferase, propionate-CoA ligase, propionyl-CoA carboxylase, methylmalonyl-CoA carboxy transferase, (S)-methylmalonyl-CoA decarboxylase, methylmalonatosemialdehyde dehydrogenase and propanal dehydrogenase . In some embodiments, class 5 glycotaxons contain enzymes associated with bile acid production (e.g., secondary bile acid production) selected from 7alpha-hydroxysteroid dehydrogenase, 12alpha-hydroxysteroid dehydrogenase, 7betahydroxysteroid dehydrogenase (NADP+), 2beta-hydroxysteroid dehydrogenase, 3beta-hydroxycholanate 3-dehydrogenase (NAD+), 3alpha-hydroxycholanate dehydrogenase (NADP+), 3beta-hydroxycholanate 3-dehydrogenase (NADP+), 3alpha-hydroxybile acid-CoA-ester 3-dehydrogenase, 3alpha-hydroxycholanate dehydrogenase (NAD+), bile acid CoA-transferase, bile acid 7alpha-dehydratase and bile acid CoA ligase. In some embodiments, class 6 glycotaxons exclude one or more enzymes associated with indole production (e.g., tryptophanase). In some embodiments, class 7 glycotaxons exclude one or more enzymes associated with p-cresol production selected from 4-hydroxyphenylacetate decarboxylase and aldehyde ferredoxin oxidoreductase.

“Isomaltooligosaccharide” or “IMOS,” as the terms are used herein, refers to a mixture of oligosaccharides with predominantly α-(1,6) linked glucose residues with a degree of polymerization (DP) ranging between 2-6, and oligosaccharides with a mixture of a-(1,6) and occasionally a-(1,4) glycosidic linkages, such as panose. In one embodiment, IMOS comprises glucosyl residues linked to maltose or isomaltose via α-(1,6) glycosidic linkages. In one embodiment, an IMOS is produced using starch as a raw material. In one embodiment, it is produced from corn starch and consists of isomaltose, isomaltriose and panose. In one embodiment, IMOS is the product of an enzymatic transfer reaction, using a combination of immobilized enzymes where starch is liquefied using α-amylase (EC 3.2.1.1) and pullulanase (EC 3.2.1.41), and, in one Second phase, the intermediate product is processed by both p-amylase (EC 3.2.1.2) and a-glucosidase (EC 3.2.1.20). Beta-amylase first hydrolyzes liquefied starch to maltose. The transglucosidase activity of α-glucosidase then produces mixtures of isomaltooligosaccharides containing oligosaccharides with glucose residues attached at both α-(1,6) and α-(1,4). In one embodiment, IMOS is an IMOS described in, or prepared by a method described in, any of references 2, or 217-219 or 12, 152, 159 or 236 of Meyer 2015. In one embodiment, IMOS is an IMOS described in, or prepared by a method described in Panesaret al.2006 or Torreset al2010, 2014 found in Diez-Municioet al.,2014. In one embodiment, IMOS is synthesized by the enzymatic hydrolysis of starch by an α-amylase or pullulanase; or a p-amylase and a-glucosidase in sequence. In one embodiment, j” [ ’ ]

IMOS is synthesized by an enzyme from A. niger, Bacillus spp., B. subtilis, stearothermophilus, T. maritime, A. carbonariousoL. mesenteroids.

As used herein, an “isolated” or “purified” glycan polymer preparation is substantially pure and free of contaminants, for example, pathogens, enzymes or otherwise unwanted biological material, or organic compounds or toxic or otherwise unwanted inorganics. In some embodiments, the pure or isolated compounds, compositions or preparations may contain traces of solvents and/or salts (such as less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% , 2%, 1%, less than 0.5% or 0.1% in w/w, w/v, v/v or mol %). The purified compounds are or preparations contain at least about 60% (in w/w, w/v, v/v or mol %), at least about 75%, at least about 90%, at least about 95% , at least about 97%, at least about 98% or at least about 99% in w/w, w/v, v/v or mol% of the compound(s) of interest. For example, a purified (substantially pure) or isolated preparation of glycan polymers is one that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9% or 100% of the glycan polymer in w/w, w/v, v/v or mol% (for example, not including any solvent, such as, for example, water, in which the glycan polymer preparation can be dissolved) and separated from the accompanying components, for example, during manufacturing, extraction/purification and/or processing (for example, so that the glycan polymer glycan is substantially free of unwanted compounds). Purity can be measured by any suitable conventional method, for example, by column chromatography (e.g., size exclusion chromatography (SEC)), thin layer chromatography (TLC), gas chromatography (GC), high-performance liquid chromatography. resolution (HPLC) or nuclear magnetic resonance spectroscopy (NMR). Purified or purity can also define a degree of sterility that is safe for administration to a human subject, for example, lacking viable infectious or toxic agents.

“Microbiome,” as used herein, refers to the genetic content of communities of microbes (“microbiota”) that live in and on a subject (e.g., a human subject), both sustainably and transiently. , including eukaryotes, archaea, bacteria and viruses (including bacterial viruses (e.g., phages)), where “genetic content” includes genomic DNA, RNA such as ribosomal RNA and messenger RNA, the epigenome, plasmids and all other types of genetic information. In some embodiments, microbiome refers specifically to the genetic content of communities of microorganisms in a niche.

“Microbiota”, as used herein, refers to the community of microorganisms that appear (sustainably or transiently) in and on a subject (e.g., a human subject), including eukaryotes, archaea, bacteria and viruses (including bacterial viruses, e.g. phages). In some embodiments, microbiota refers specifically to the microbial community in a niche.

“Pathobionts” or “(opportunistic) pathogens,” as used herein, refer to symbiotic organisms capable of causing disease only when certain genetic and/or environmental conditions are present in a subject (e.g., a human subject). ).

As used herein, the term “pathogen” (e.g., “pathogenic bacteria”) refers to a substance, microorganism, or condition that has the capacity to cause disease. In certain contexts, pathogens also include microbes (e.g. bacteria) that are associated with a disease or condition but for which a causal (direct) relationship has not or has not yet been established. As used herein, the term “pathogens” refers to viruses, parasites, and bacteria or other pathogens that can cause infections in a subject, for example, a human.

As used herein, a “pharmaceutical composition” is a composition or preparation that has pharmacological activity or other direct effect in the mitigation, treatment or prevention of diseases, and/or a finished dosage form or formulation of the same and is for human use. A pharmaceutical composition is typically produced under good manufacturing practice (GMP) conditions. Pharmaceutical compositions may be sterile or non-sterile. If non-sterile, such pharmaceutical compositions typically meet the specifications and microbiological criteria for non-sterile pharmaceutical products described in the US Pharmacopeia (USP) or the European Pharmacopeia (EP). The pharmaceutical compositions may further comprise or may be co-administered with additional active agents, such as, for example, additional therapeutic agents. The pharmaceutical compositions may also comprise, for example, additional therapeutic agents, polyphenols, prebiotic substances, probiotic bacteria, excipients, solvents, pharmaceutically acceptable carriers or any combination thereof. Any glycan polymer preparation described herein can be formulated as a pharmaceutical composition.

The term “subject” (in some cases “patient”) as used herein refers to any human subject. The term does not indicate any particular age or gender. Subjects may include pregnant women. Subjects may include a newborn (a premature newborn, a full-term newborn), an infant up to one year of age, young children (e.g., 1 year to 12 years), adolescents (e.g., 13 to 19 years), adults (for example, 20 to 64 years) and older adults (65 years and older). A subject does not include an agricultural animal, for example, farm animals or livestock, for example, cattle, horses, sheep, pigs, chickens, etc.

A “substantial decrease” as used herein (e.g., with respect to a biomarker or metabolite) is a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%. 70%, 80%, 90%. %, 95%, 97%, 98%, 99%, 99.9% or 100%.

A “substantial increase” as used herein (e.g., with respect to a biomarker or metabolite) is an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%. 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800% , 850%, 900%, 950%, 1000% or more than 1000%.

The term "substrate", as used herein in relation to the terms glycosidase enzyme and/or glycosidase enzyme molecule, refers to a glycan polymer that is the product of, or has the structure of, a polymer of glycan prepared by a glycosidase enzyme molecule; and is the substrate of a glycosidase enzyme, for example, a glycosidase expressed in a human intestinal microbe. In some embodiments, the glycosidase enzyme molecule, under appropriate reaction conditions, catalyzes the polymerization of glycan subunits to form the substrate, and the glycosidase enzyme, under appropriate reaction conditions, cleaves a bond between glycan subunits (In In some embodiments, the same bond formed by the glycosidase enzyme molecule) of the substrate. In one embodiment, the glycosidase enzyme molecule and the glycosidase enzyme have the same primary amino acid sequence, for example, they are the same enzyme. In some embodiments, the substrate has one or more of the following properties:

i) is sufficiently similar to a naturally occurring substrate of the glycosidase enzyme such that the turnover rate for the substrate and the glycosidase enzyme is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 99% of that of at least one naturally occurring substrate of the glycosidase enzyme. The turnover rate may be expressed, for example, in terms of glycosidic bonds cleaved per unit of time, for example, per minute or hour, or rate of depolymerization of the glycan polymer per unit of time, for example, hour or minute; ii) its binding constant for the glycosidase enzyme is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 99% of that of at least one substrate that occurs naturally from the glycosidase enzyme, and in embodiments is no more than 1, 2, 3, 4, 5, 10, 50 or 100 times greater than that of at least one naturally occurring substrate of the glycosidase enzyme; and iii) their binding motif for the glycosidase enzyme, their binding motif for the glycosidase enzyme molecule and at least one naturally occurring substrate of the glycosidase enzyme share one or more than one specific glycan subunit, e.g. , a specific sugar dimer, a specific sugar branch point, a specific alpha or beta configuration, a specific regiochemistry, e.g. a 1,2-, 1,3-, 1,4-, 1,5- linkage or 1.6; and iv) the substrate promotes the growth or metabolism of a human intestinal microbe that expresses the enzyme molecule.

“Synthetic,” as used herein, refers to an artificial compound or preparation, such as a glycan polymer preparation, that does not occur naturally. In one embodiment, a non-enzymatic polymeric catalyst described herein is used to synthesize the glycans of the preparation under suitable reaction conditions, for example, by a polymerization reaction that creates oligomers from individual glycan subunits that are added to the reaction. In some embodiments, the non-enzymatic polymeric catalyst acts as a hydrolysis agent and can cleave glycosidic bonds. In other embodiments, the non-enzymatic polymeric catalyst may form glycosidic bonds. In one embodiment, a glycosidase enzyme molecule described herein is used to synthesize the glycans of the preparation under suitable reaction conditions, for example, by a polymerization reaction that creates oligomers from individual glycan subunits that are added to the reaction. In some embodiments, the glycosidase enzyme molecule acts as a hydrolysis agent and can cleave glycosidic bonds. In other embodiments, the glycosidase enzyme molecule may form glycosidic bonds. In one embodiment, solid phase oligosaccharide synthesis is used to synthesize the glycans of the preparation under suitable reaction conditions, for example, by a polymerization reaction that creates oligomers from individual glycan subunits that are added to the reaction. Synthetic glycan polymer preparations may also include glycan polymers that are not isolated from a natural source of oligo or polysaccharides. It should be understood that, although the glycan polymer preparation is not isolated from a natural oligo or polysaccharide source, the glycan subunits that constitute the glycan polymer can and often are isolated from natural oligo or polysaccharide sources, including those listed in this document, or are synthesized de novo.

The terms "treat" and "treatment", as used herein, refer to the administration of an agent or composition to a subject (for example, a symptomatic subject afflicted with an adverse condition, disorder or disease) to affect a reduction in the severity and/or frequency of a symptom, eliminate a symptom and/or its underlying cause, and/or facilitate the improvement or repair of damage, and/or prevent an adverse condition, disorder or disease in a subject asymptomatic (e.g., a human subject) who is susceptible to a particular adverse condition, disorder or disease, or who is suspected of developing or is at risk of developing the condition, disorder or disease.

A “therapeutic nutrition product” is a food product that provides a therapeutic effect, either when administered alone or in combination with a second therapy (for example, a drug therapy), in which case it provides an additive or synergistic therapeutic effect. or alleviates or reduces negative effects of the second therapy (for example, reduction of side effects). A therapeutic nutrition product is part of a recommended diet (for example, by a physician or dietician or other expert in dietetics, human nutrition) and the regulation of a diet (for example, based on the medical condition and individual needs of a subject).

"Xylooligosaccharide" or "XOS", as the terms are used herein, refer to sugar oligomers of xylose units linked by p-(1,4). The number of xylose residues varies from 2 to 10, but they mainly consist of xylobiose, xylotriose, and xylotetraose. Arabinofuranosyl, glucopyranosyluronic acid or its 4-O-methyl derivative (2- or 3-acetyl or phenolic substituents) may also be present and result in branched XOS. In one embodiment, XOS is primarily linear (P-(1,4)-linked of lignocellulosic materials. In one embodiment, xylan is enzymatically hydrolyzed to xylooligosaccharides by an endo-p-1,4-xylanase (EC 3.2.1.8) or by beta-xylosidase (EC 3.2.1.9). Xos through enzymatic xilanos degradation, for example, through an endo-p-1,4-Xilanas p-coumaric In one embodiment, XOS is an described in or prepared by a method described in Casci and Rostal, 2006, found in Diez-Municioet al.,2014. In one embodiment, XOS is synthesized by a xylanase from any of T. reesei, T. harzianu, T. viride, T. koningii, T. longibrachiatum, P. chyrosporium,G. trabeumoA. oryzae.

When the term “comprising” is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term “consisting of” is considered a preferred embodiment of the term “comprising.” If a group is hereinafter defined to comprise at least a certain number of embodiments, this should also be understood to mean a group preferably consisting solely of these embodiments.

When an indefinite or definite article is used to refer to a singular noun, for example, “a,” “an,” or “the,” this includes a plural of that noun unless something else is specifically indicated.

Claims and disclosure that refer to methods of treatment or diagnosis are considered an equivalent disclosure of embodiments and claims to “compound, composition, product, etc. for use in ...” or “use of a compound, composition, product, etc. in the manufacture of a medicine, pharmaceutical composition, diagnostic composition, etc. for..." and indicates that such compounds, compositions, products, etc. They are going to be used in diagnostic or therapeutic methods that can be put into practice on the human or animal body. If an embodiment or claim therefore refers to a “method of treatment by administering a compound to a human or animal suspected of suffering from a disease”, this is also considered a disclosure of a “use of a compound in the manufacture of a medicinal product for the treatment of a human being or animal suspected of suffering from a disease” or “a compound for use in the treatment of a human being or animal suspected of suffering from a disease” . As an example: a reference to a method of treating a subject who has a disease or disorder associated with an undesirable level of a metabolite (e.g., a short chain fatty acid (SCFA), ammonia, trimethylamine (TMA), N -trimethylamine oxide (TMAO), a uremic solute, lipopolysaccharide (LPS) or a bile acid) by administering an amount of a glycan polymer preparation is considered a disclosure of (i) a glycan polymer preparation for its use in the treatment of a subject who has a disease or disorder associated with an unwanted level of a metabolite (for example, a short chain fatty acid (SCFA), ammonia, trimethylamine (TMA), trimethylamine N-oxide (TMAO ), a uremic solute, lipopolysaccharide (LPS) or a bile acid) or (ii) use of a glycan composition in the manufacture of a medicament to treat a subject having a disease or disorder associated with an unwanted level of a metabolite (for example, a short chain fatty acid (SCFA), ammonia, trimethylamine (TMA), trimethylamine N-oxide (TMAO), a uremic solute, lipopolysaccharide (LPS), or a bile acid).

Whenever reference is made to a method of treating an individual or a population of individuals by administering, for example, a glycan composition as a reference, in a preferred embodiment, it contemplates an analytical, diagnostic and similar step in the course of such treatment that can help determine, for example, whether an individual or population will be susceptible to a certain treatment due to their microbiome composition, whether it was successful, etc. By way of example: a reference to a method of treating a subject who has a disease or disorder associated with an undesirable level of a metabolite (e.g., a short chain fatty acid (SCFA), ammonia, trimethylamine (TMA) , trimethylamine N-oxide (TMAO), a uremic solute, lipopolysaccharide (LPS) or a bile acid) by administering an amount of a glycan polymer preparation is also considered a disclosure of such a method, wherein, in a preferred embodiment the subject will, for example, (i) be tested initially to determine the nature and level of the metabolite before starting treatment, (ii) the composition of his microbiome will be tested to adapt the administration of the glycan composition to the composition of the microbial glycosidase enzyme in the intestine, (iii) will be tested in the course of treatment to monitor the effect of administration of the glycan composition on the level of metabolite, etc.

The terms "obtainable by", "produceable by" or the like are used to indicate that a claim or embodiment refers to a compound, composition, product, etc. per se, that is, that the compound, composition, product, etc. can be obtained or produced by a method that is described for the manufacture of the compound, composition, product, etc., but that the compound, composition, product, etc. It can also be obtained or produced by methods other than that described. The terms “obtained by”, “produced by” or the like indicate that the compound, composition, product is obtained or produced by a specific method mentioned. It should be understood that the terms "obtainable by", "produceable by" and the like also disclose the terms "obtained by", "produced by" and the like as a preferred embodiment of "obtainable by", "producible by" and the like.

It should be further understood that the present disclosure, as preferred embodiments, also discloses how the individual aspects and embodiments described herein may be combined. For example, Table 3 reports an association between metabolites and phyla and strains, while Table 5 reports an association between metabolites and diseases. Therefore, the person skilled in the art will consider this information together and understand which microorganisms must be influenced to, for example, reduce the level of a metabolite in order to treat a certain disease.

As used herein, “homology” and “sequence identity” (used interchangeably herein) are measures of the similarity of a sequence (e.g., amino acid sequences or nucleic acid sequences) to another sequence. . Calculations of “homology” or “sequence identity” between two sequences (the terms are used interchangeably herein) are performed as follows. The sequences are aligned for optimal comparison purposes (for example, gaps may be introduced in one or both of a first and second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences may be discarded for comparison purposes). The optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a fact extension penalty of 4, and a displacement gap penalty of 5 frame. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, amino acid “identity”). or nucleic acid is equivalent to “homology” of amino acid or nucleic acid). The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences.

Preparation methods of glycan polymers

The following aspects and embodiments for preparing a glycan polymer are for illustrative purposes. A glycan polymer preparation can be produced using any method known in the art. The glycan polymer compositions may comprise the glycans described herein, dietary fibers, such as, for example, FOS (fructo-oligosaccharide), other sugars (for example, monomers, dimers, such as, for example, lactulose) and sugar alcohols, and optionally other components, such as, for example, polyphenols, fatty acids, peptides, micronutrients, etc., such as those described in WO 2016/172658, “MICROBIOME REGULATORS AND RELATED USES THEREOF”, and microbes, such as bacteria.

The glycan preparations described in WO 2016/122889 “GLYCAN THERAPEUTICS AND RELATED METHODS THEREOF” and WO 2016/172657, “GLYCAN THERAPEUTICS AND METHODS OF TREATMENT”, are suitable for use in the methods and compositions described in This document.

Preparations comprising glycan polymers can be generated using a non-enzymatic catalyst, for example, the polymeric catalyst described in WO 2012/118767, “POLYMERIC ACID CATALYSTS AND USES THEREOF” or by other suitable methods. Other acid catalysts (eg, solid catalysts) may be used. Methods for preparing the polymeric and solid-supported catalysts described herein can be found in WO 2014/031956, “POLYMERIC AND SOLID-SUPPORTED CATALYSTS, AND METHODS OF DIGESTING CELLULOSIC MATERIALS USING SUCH CATALYSTS.” Glycans generated, for example, using the catalyst, for example as described in WO 2016/007778, “OLIGOSACCHARIDE COMPOSITIONS AND METHODS FOR PRODUCING THEREOF” are suitable for the methods and compositions described herein.

In some embodiments, glycan polymers are prepared using solid phase oligosaccharide synthesis, for example, using a variety of protecting groups to achieve glycan synthesis. Exemplary methods are described in “Solid-Phase Oligosaccharide Synthesis and Combinatorial Carbohydrate Libraries”, Peter H. Seeberger and Wilm-Christian Haase, American Chemical Society, 2000; and “Opportunities and challenges in synthetic oligosaccharide and glycoconjugate research”, Thomas J. Boltjeet al., Nat Chem. November 1, 2009; 1(8): 611-622.

In some embodiments, glycan polymers can be synthesized using an enzymatic catalyst (e.g., a glycosidase or glycosyltransferase, either isolated or expressed in bacteria), as described herein, to synthesize the glycans via a polymerization reaction. which creates oligomers from individual glycan subunits that are added to the reaction. Exemplary methods are described in “Synthesis and Purification of Galacto-Oligosaccharides: State of the Art”, Carlos Veraet al., World J. Microbiol Biotechnol. 2016; 32:197; “Synthesis of Novel Bioactive Lactose-Derived Oligosaccharides by Microbial Glycoside Hydrolases”, Marina Diez-Municioet al., Microbial Biotechnol. 2014; 7(4), 315-331; and “Methods of Improving Enzymatic Trans-Glycosiltion for Synthesis of Human Milk Oligosaccharide Biomimetics”, Birgitte Zeuneret al.,J. Agric. Food Chem. 2014, 62, 9615-9631, document WO 2005/003329 “NOVEL GALACTOOLIGOSACCHARIDE COMPOSITION AND THE PREPARATION THEREOF”.

In some embodiments, glycan preparations can be prepared using glycan polymers, such as starch and other fibers, such as dietary fibers (such as those described herein) and subjecting them to a catalyst (for example, an acid catalyst, a solid or polymeric catalyst, an enzymatic catalyst) to change one or more properties of the glycan (or fiber), for example, the degree of polymerization (for example, depolymerization), the degree of branching (for example, debranching) or the distribution of glycosidic bonds (for example, by adding new types of glycosidic bonds or removing existing bonds). An exemplary method for corn syrup is described in US Patent Publication No. 2016/0007642, Example 101. Other methods may be used, such as those used for the preparation of resistant starch (e.g., described in M.G. Sajilataet al., “Resistant Starch - A Review,” Comprehensive Reviews in Food Science and Food Safety - vol. 5, 2006, and US Patent Publication No. 2006/0257977, “Slowly digestible starch”), such as, for example, heat treatment, enzymatic treatment, chemical treatment, or a combination thereof, to produce the glycan preparations described herein.

Glycan subunits

The following aspects and embodiments of glycan subunits are for illustrative purposes and provide information regarding the preparation of glycan polymer for use in the method of the invention. The present invention presents methods of preparation or methods of manufacturing a preparation of a glycan polymer that is a substrate for an intestinal microbe (for example, a human intestinal microbe). Starting materials for such methods are glycan subunits comprising sugar monomers (e.g., monosaccharides), sugar dimers (e.g., disaccharides), sugar trimers (e.g., trisaccharides), or combinations thereof.

The starting material may comprise a furanose sugar or a pyranose sugar. In some embodiments, the starting material comprises a tetrose, a pentose, a hexose or a heptose. In some embodiments, the starting material comprises glucose, galactose, arabinose, mannose, fructose, xylose, fucose and rhamnose. The glycan subunit starting materials may be in either their L or D form, in the alpha or beta configuration and/or a deoxy form, where applicable, and any combination thereof. The glycan subunits used in the methods described herein may include a monosaccharide, such as a C5 monosaccharide or a C6 monosaccharide. In some embodiments, the monosaccharide is a C5 monosaccharide. In some embodiments, the monosaccharide is a C6 monosaccharide. The glycan subunits may include a disaccharide, such as a disaccharide comprising a C5 monosaccharide or a C6 monosaccharide. In some embodiments, the disaccharide comprises a C5 monosaccharide. In some embodiments, the disaccharide comprises two C5 monosaccharides. In some embodiments, the disaccharide comprises a C6 monosaccharide. In some embodiments, the disaccharide comprises two C6 monosaccharides. In some embodiments, the disaccharide comprises one of a C5 monosaccharide and one of a C6 monosaccharide. The glycan subunit starting material used herein may be a monosaccharide selected from glycoaldehyde, glycolaldehyde, glyceraldehyde, dihydroxyacetone, erythrose, threose, erythulose, arabinose, lyxose, ribose, xylose, ribulose, xylulose, allose, altrose, galactose. , glucose, gulose, idose, mannose, thallose, fructose, psychose, sorbose, tagatose, fucose, fuculose, rhamnose, mannoheptulose, sedoheptulose, neuraminic acid, N-acetylneuraminic acid, N-acetylgalactosamine, N-acetylglucosamine, fructosamine, galactosamine, glucosamine , sorbitol, glycerol, erythritol, threitol, arabitol, xylitol, mannitol, sorbitol, galactitol, fucitol and lactic acid.

The glycan subunit starting material used herein may be a disaccharide or a larger subunit selected from acarviosin, N-acetyl-lactosamine, allolactose, cellobiose, chitobiose, glactose-alpha-1,3-galactose, gentiobiose , isomaltose, isomaltose, isomaltulose, kojibiose, lactitol, lactobionic acid, lactose, lactulose, laminaribose, maltitol, maltose, mannobiose, melibiose, melibiulose, neohesperidose, nigerose, robinose, rutinose, sambubiose, sophorose, sucralose, sucrose, sucrose acetate isobutyrate , sucrose octaacetate, trehalose, turanose, vicianose and xylobiose

In some embodiments, the glycan subunit is an inactivated glycan subunit. In some embodiments, the glycan subunit is an activated glycan subunit, e.g., activated with a nucleoside, nucleotide (e.g., UTP, UDP, UMP, GTP, GDP, GMP, ATP, ADP, AMP, CTP, CDP, CMP) or phosphate group. In some embodiments, the glycan subunit is a UDP sugar or a UMP sugar.

In some embodiments, the glycan subunit is substituted or derivatized with an acetyl group, acetate ester, sulfate half-ester, phosphate ester, or a cyclic pyruvyl acetal group, or has otherwise been derivatized into, for example, one or more hydroxyl groups or amine groups.

In some embodiments, the glycan subunit comprises an aminosugar, deoxysugar, iminosugar, sugar acid, or sugar alcohol. Exemplary amino sugars include acarbose, N-acetylmannosamine, N-acetylmuramic acid, N-acetylneuraminic acid, N-acetylthalosaminuronic acid, arabinopyranosyl-N-methyl-N-nitrosourea, D-fructose-L-histidine, N-glycolineuraminic acid , ketosamine, kidamycin, mannosamine, 1B-methylselene-N-acetyl-D-galactosamine, muramic acid, muramyl dipeptide, phosphoribosylamine, PUGNAc, sialyl-Lewis A, sialyl-Lewis , aspartylglucosamine, bacilthiol, daunosamine, desosamine, fructosamine, galactosamine, glucosamine, meglumine and perosamine. Exemplary deoxysugars include 1-5-ahydroglucitol, cladinose, colitose, 2-deoxy-D-glucose, 3-deoxyglucasone, deoxyribose, dideoxynucleotide, digitalose, fludeoxyglucose, sarmentose and sulfoquinovose. Exemplary iminosugars include castanospermine, 1-deoxynojirimycin, iminosugar, miglitol, miglustat and swainsonine. Exemplary sugar acids include N-acetylneuraminic acid, N-acetylthalosamnuronic acid, aldaric acid, aldonic acid, 3-deoxy-D-manno-oct-2-ulosonic acid, glucuronic acid, glucosaminouronic acid, glyceric acid, N-glycolylneuraminic acid, iduronic acid, acid, isosacharinic acid, pangamic acid, sialic acid, threonic acid, ulosonic acid, uronic acid, xylonic acid, gluconic acid, ascorbic acid, ketodeoxyoctulosonic acid, galacturonic acid, galactosaminuronic acid, mannuronic acid, mannosaminuronic acid , tartaric acid, mucic acid, saccharic acid, lactic acid, oxalic acid, succinic acid, hexanoic acid, fumaric acid, maleic acid, butyric acid, citric acid, glucosamine acid, malic acid, succinamic acid, sebacic acid and capric acid. Exemplary sugar alcohols include methanol, ethylene glycol, glycerol, erythritol, threitol, arabitol, ribitol, xylitol, mannitol, sorbitol, galactitol, iditol, volemitol, fucitol, inositol, maltotritol, maltotetraitol and polyglycitol. In some embodiments, the glycan subunit starting material is a salt (e.g., a pharmaceutically acceptable salt), such as, for example, a salt of hydrochloride, hydroiodide, hydrobromide, phosphate, sulfate, methanesulfate, acetate, formate, tartrate, malate, citrate, succinate, lactate, gluconate, pyruvate, fumarate, propionate, aspartate, glutamate, benzoate, ascorbate.

A glycan subunit used in a method described herein can be obtained from any commercially known source or produced according to any method known in the art. In some embodiments, hydrolysis may be used to generate the constituent monosaccharides or oligosaccharides that are suitable for producing the glycans described herein. Glycan units, such as, for example, monosaccharides, can exist in many different forms, for example, conformers, cyclic forms, acyclic forms, stereoisomers, tautomers, anomers and isomers.

Preparation of glycan polymers using a non-enzymatic polymer catalyst

The following aspects and embodiments for preparing glycan polymers using a non-enzymatic polymer catalyst are for illustrative purposes.

Reaction conditions

In some embodiments, the glycan unit and the catalyst (e.g., polymeric catalyst or solid supported catalyst) are allowed to react for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 16 hours, at least 24 hours, at least 36 hours, or at least 48 hours; or between 1-24 hours, between 2-12 hours, between 3-6 hours, between 1-96 hours, between 12-72 hours, or between 12-48 hours.

In some embodiments, the degree of polymerization of the one or more oligosaccharides produced according to the methods described herein can be regulated by reaction time. For example, in some embodiments, the degree of polymerization of the one or more oligosaccharides increases as the reaction time increases, while, in other embodiments, the degree of polymerization of the one or more oligosaccharides decreases as the reaction time decreases.

reaction temperature

In some embodiments, the reaction temperature is maintained in the range of about 25°C to about 150°C. In certain embodiments, the temperature is from about 30°C to about 125°C, from about 60°C to about 120°C, from about 80°C to about 115°C, from about 90°C to about 110°C. C, from about 95°C to about 105°C or from about 100°C to 110°C.

Number of glycan units

The amount of the glycan unit used in the methods described herein relative to the amount of solvent used can affect the reaction rate and yield. The amount of the glycan unit used can be characterized by the dry solids content. In certain embodiments, dry solids content refers to the total solids of a suspension as a percentage on a dry weight basis. In some embodiments, the dry solids content of the glycan unit is between about 5% by weight and about 95% by weight, between about 10% by weight and about 80% by weight, between about 15% by weight and about 15% by weight. % by weight and about 75% by weight or between about 15% by weight and about 50% by weight.

Catalyst quantity

The amount of the catalyst used in the methods described herein may depend on various factors including, for example, the selection of the type of glycan unit, the concentration of the glycan unit, and the reaction conditions (e.g., temperature, time and pH). In some embodiments, the weight ratio of the catalyst to the glycan unit is from about 0.01 g/g to about 50 g/g, from about 0.01 g/g to about 5 g/g, from about 0. 05 g/g to about 1.0 g/g, about 0.05 g/g to about 0.5 g/g, about 0.05 g/g to about 0.2 g/g or about 0.1 g/g g to approximately 0.2 g/g.

Solvent

In certain embodiments, the methods of using the catalyst are carried out in an aqueous environment. A suitable aqueous solvent is water, which can be obtained from various sources. Generally, water sources with lower concentrations of ionic species (e.g., sodium, phosphorus, ammonium, or magnesium salts) are preferable, since such ionic species can reduce catalyst effectiveness. In some embodiments, when the aqueous solvent is water, the water has a resistivity of at least 0.1 megohm-centimeters, at least 1 megohm-centimeters, at least 2 megohms-centimeters, at least 5 megohms-centimeters, or at least 5 megohms-centimeters. minus 10 megohm-centimeters.

water content

Furthermore, as the dehydration reaction of the methods progresses, water is produced with each coupling of the one or more glycan units. In certain embodiments, the methods described herein may further include monitoring the amount of water present in the reaction mixture and/or the ratio of water to monomer or catalyst over a period of time. In some embodiments, the method further includes removing at least a portion of the water produced in the reaction mixture (e.g., removing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70% %, 80%, 90%, 95%, 97%, 99% or 100%, such as by vacuum filtration). However, it should be understood that the amount of water relative to monomer can be adjusted based on the reaction conditions and the specific catalyst used.

Any method known in the art can be used to remove water in the reaction mixture, including, for example, by vacuum filtration, vacuum distillation, heating and/or evaporation. In some embodiments, the method comprises including water in the reaction mixture.

In some aspects, methods of producing an oligosaccharide composition are provided herein: combining a glycan unit and a catalyst having acidic and ionic moieties to form a reaction mixture, wherein water is produced in the reaction mixture ; and removing at least a portion of the water produced in the reaction mixture. In certain variations, at least a portion of the water is removed to maintain a water content in the reaction mixture of less than 99%, less than 90%, less than 80%, less than 70%, less than 60%, less 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 1% by weight.

In some embodiments, the degree of polymerization of the one or more oligosaccharides produced according to the methods described herein can be regulated by adjusting or controlling the concentration of water present in the reaction mixture. For example, in some embodiments, the degree of polymerization of the one or more oligosaccharides increases with decreasing water concentration, while, in other embodiments, the degree of polymerization of the one or more oligosaccharides decreases with increasing water concentration. In some embodiments, the water content of the reaction is adjusted during the reaction to regulate the degree of polymerization of the one or more oligosaccharides produced.

In one example, one or more mono-, di-, tri-, or other oligosaccharides may be added to a round-bottom flask equipped with an overhead stirrer and a jacketed short path condenser along with 1-50% (1-10%, 1 -20%, 1-30%, 1-40%, 1-60%, 1-70%) by dry weight of one or more of the catalysts described herein. Water or other compatible solvent (0.1-5 equiv., 1-5 equiv., 1-4 equiv., 0.1 4 equiv.) may be added to the dry mixture and the suspension may be combined at a slow rate (e.g. , 10-100 rpm, 50 200 rpm, 100-200 rpm) using a paddle size that matches as closely as possible the contours of the selected round-bottom flask. The mixture is heated to 70-180 °C (70-160 °C, 75-165 °C, 80-160 °C) under a vacuum pressure of 10-1000 mbar. The reaction can be stirred for 30 minutes to 6 hours, constantly removing reaction water. The progress of the reaction can be monitored by HPLC. The solid mass obtained through the process can be dissolved in a sufficient volume of water to create a solution of approximately 50 Brix (grams of sugar per 100 g of solution). Once dissolution is complete, the solid catalyst can be removed by filtration and the oligomer solution can be concentrated to about 50-75 Brix, for example, by rotary evaporation. Optionally, an organic solvent can be used and the water-immiscible solvents can be removed by two-phase extraction and the water-miscible solvents can be removed, for example, by rotary evaporation concomitant to the concentration step.

Preparation of glycan polymers using a glycosidase enzyme molecule

The following aspects and embodiments for preparing a glycan polymer using a glycosidase enzyme molecule are for illustrative purposes.

Reaction conditions

A glycan polymer produced using the methods described herein can be generated by condensation (e.g., reverse hydrolysis) and/or transglycosylation of a glycosidic bond catalyzed by a glycosidase enzyme molecule (e.g., a hydrolase, transferase or lyase). . In some embodiments, a characteristic of a glycan polymer produced according to the methods described herein can be regulated by a reaction condition, for example, reaction time, reaction temperature, concentration or amount of a glycan subunit, concentration or amount of a glycosidase enzyme molecule, solvent or an additional processing step, for example, as described herein. In some embodiments, the reaction conditions of the methods described herein reflect physiological conditions, for example, pH between 5 and 7.5 and a temperature between 35 ° C and 60 ° C. In some embodiments, the reaction conditions of a method described herein deviate from physiological conditions.

Reaction time

In some embodiments, the glycosidase enzyme molecule and a starting material (e.g., a glycan subunit) are allowed to react for at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 16 hours, at least 24 hours, at least 36 hours or at least 48 hours. In some embodiments, the glycosidase enzyme molecule and a starting material (e.g., a glycan subunit) are allowed to react between 1-24 hours, between 2-12 hours, between 3-6 hours, between 1-96 hours , between 12-72 hours or between 12-48 hours. In some embodiments, the degree of polymerization (DP) of a glycan polymer produced according to the methods described herein can be regulated by reaction time. For example, in some embodiments, the degree of polymerization of a glycan polymer increases with increasing reaction time, while, in other embodiments, the degree of polymerization of a glycan polymer decreases with decreasing reaction time.

reaction temperature

In some embodiments, the reaction temperature is maintained in the range of about 4°C to about 150°C. In certain embodiments, the temperature is from about 4°C to about 30°C, from about 4°C to about 125°C, from about 30°C to about 125°C, from about 60°C to about 120°C. C, from about 80°C to about 115°C, from about 90°C to about 110°C, from about 95°C to about 105°C or from about 100°C to 110°C. In some embodiments, the reaction temperature is room temperature (e.g., about 25°C). In some embodiments, the reaction temperature is physiological temperature (e.g., about 30°C). In some embodiments, the reaction temperature is about 60°C.

In some embodiments, the reaction is substantially slowed or stopped after a period of time by increasing the temperature, for example, through denaturation of the enzyme. In some embodiments, the reaction is substantially slowed or stopped by increasing the temperature to greater than about 45°C, about 50°C, about 60°C, about 70°C, about 80°C, about 90°C, about 100°C. C, approximately 110 °C or higher.Concentration or amount of a glycan subunit

The concentration or amount of a glycan subunit used in the methods described herein relative to the amount of solvent used can affect the reaction rate and yield. In some embodiments, the concentration or amount of a glycan subunit is about 10 mg/ml, about 25 mg/ml, about 50 mg/ml, about 75 mg/ml, about 100 mg/ml, about 200 mg/ml. , about 300 mg/ml, about 400 mg/ml, about 500 mg/ml, about 750 mg/ml, about 1 g/ml, or more.

The amount of glycan subunit used can be characterized by the dry solids content. In certain embodiments, dry solids content refers to the total solids of a suspension as a percentage on a dry weight basis. In some embodiments, the dry solids content of the glycan subunit is between about 5% by weight and about 95% by weight, between about 10% by weight and about 80% by weight, between about 15 % by weight and approximately 75% by weight or between approximately 15% by weight and approximately 50% by weight.

Concentration or amount of a glycosidase enzyme molecule

The concentration or amount of the glycan enzyme molecule used in the methods described herein may depend on several factors including, for example, the selection of the type of glycan subunit, the concentration of the glycan subunit, and the conditions of reaction (e.g. temperature, time and pH). In some embodiments, the concentration or amount of the glycosidase enzyme molecule is about 0.1 U/ml, about 0.5 U/ml, about 1 U/ml, about 5 U/ml, about 10 U/ml, approximately 25 U/ml, approximately 50 U/ml or greater. In some embodiments, the concentration or amount of the glycosidase enzyme molecule is between 0.1-5 U/ml, between 1-25 U/ml or 1-50 U/ml. In some embodiments, the weight ratio of the glycosidase enzyme molecule to the glycan subunit is from about 0.01 g/g to about 50 g/g, from about 0.01 g/g to about 5 g/g, from about 0.05 g/g to about 1.0 g/g, from about 0.05 g/g to about 0.5 g/g, from about 0.05 g/g to about 0.2 g/g or from about 0 .1 g/g to approximately 0.2 g/g.

Solvent

In some embodiments, the reaction solvent is a biocompatible solvent. In certain embodiments, the reaction solvent is an aqueous solvent, for example, water or a mixture of water. In some embodiments, the reaction solvent is water or a mixture of water and a miscible solvent such as acetone, ethanol, isopropanol, polyethylene glycol, t-butanol or other solvent. In some embodiments, the reaction solvent is an organic solvent (e.g., a pure organic solvent).

A solvent may be added to the reaction mixture in order to increase the reaction rate or the overall yield of the reaction, for example, through increasing the accessibility of a glycosidase enzyme molecule to a glycan subunit. Exemplary solvents include an organic solvent such as DMSO and toluene.

Components of the addition reaction

The reaction mixture may comprise an additional component such as a salt, a detergent, a metal, a chelator, an acid, a base, a cofactor, a coenzyme, a vitamin, an amino acid, a prosthetic group, a nucleoside, a nucleotide. , or any combination thereof. In some embodiments, the reaction mixture comprises a cofactor or coenzyme such as NAD+, NADH, NADP+, NADPH, FAD, FADH, coenzyme A, biotin, pyridoxal phosphate or methylcobalamin. In some embodiments, the inclusion of an additional component improves the reaction yield, the enzyme turnover rate, the stability of the enzyme, the stability of the glycan subunit, the stability of the glycan polymer, or any combination of the same.

Glycosidase enzymes

Described herein are methods of preparing glycan polymer preparations that are substrates for a glycosidase enzyme, for example, a glycosidase enzyme present in a human intestinal microbe. In their natural environments, for example, expressed by an intestinal bacteria in the intestine of a subject, glycosidases use glycan polymers as substrates, for example, they recognize specific glycan polymers and hydrolyze glycosidic bonds in the glycan polymer. This hydrolysis can lead to the release of monomers or dimers of the glycan polymer, a shortening of the glycan polymer and/or debranching (e.g., the removal of a glycosidic branch point from the glycan polymer). The action of glycosidase provides the microbe with glycan degradation products that it can convert into energy. This process is known as glycan fermentation. Many glycosidases are specific, for example they have recognition motifs at the end of the glycan chain (e.g. exoglycosidases) or within (e.g. endoglycosidases), they can recognize specific sugars or combinations of sugars (e.g. glu-glu or glu-gal) and may also be selective in stereochemistry and/or regiochemistry (e.g., recognition of alpha versus beta glycosidic bonds, and/or 1->2 versus 1->3 versus 1->6 bonds). Some glycosidase enzymes are more promiscuous, having a wider variety of glycan polymer substrates.

In artificial environments and under suitable conditions, glycosidase enzymes can produce glycan polymers, for example by condensation reaction and/or transglycosylation reactions. The glycan polymers produced may have a higher degree of polymerization than the inputs, they may present branching and stereochemical and/or regiochemical variety (with respect to alpha-beta glycosidic bonds and unions). Exemplary glycosidase enzymes include hydrolases, transferases or lyases. A glycosidase enzyme can be characterized in various ways, such as by its sequence, size or function. In some embodiments, a glycosidase enzyme is associated with a bacteria of a particular taxon. In some embodiments, a glycosidase enzyme has a CAZy family designation (i.e., the family designation provided by the Carbohydrate Active enZYme database (http://www.cazy.org/)), e.g., glycosylhydrolase family (GH) or glycosyltransferase (GT) family, based on the analysis of genomic, structural and biochemical information. In some embodiments, a glycosidase enzyme (e.g., a naturally occurring glycosidase enzyme, e.g., expressed by an intestinal microbe) is a glycosidase enzyme molecule (e.g., a glycosidase used in methods of preparing a glycan polymer described herein). In some embodiments, the glycosidase enzyme molecule is 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the glycosidase enzyme (e.g., by DNA sequence, RNA sequence or amino acid sequence). In other embodiments, the glycosidase enzyme molecule comprises a deletion, additional sequence, point mutation, conservative or non-conservative amino acid changes, codon optimization, purification tags, folding/stability promoting mutations, etc. compared to the enzyme glycosidase. In some embodiments, the glycosidase enzyme is a member of a GH CAZY family. In some embodiments, the glycosidase enzyme is a member of a GT CAZY family. In some embodiments, the glycosidase enzyme molecule is related to (or derivatized from) the glycosidase enzyme having one or more sequence modifications (e.g., DNA, RNA, or amino acid sequence), such as those described herein.

In some embodiments, the glycosidase enzyme or glycosidase enzyme molecule is present in a human intestinal microbe. The glycosidase enzyme can be isolated from the microbe. In some embodiments, the glycosidases are present in a microbial supernatant, present in a microbial extract, present in a mass of microbial cells, or are isolated to essential purity (e.g., essentially pure enzyme fraction). In some embodiments, the glycosidase enzyme is derived from human intestinal microbes. In some embodiments, the glycosidase enzyme is derived from a yeast, fungus, or bacteria. In one embodiment, the glycosidase enzyme comes from a bacteria, such as a human intestinal bacteria. In some embodiments, the bacterial taxon is one of Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, Spirochaetes, Synergistetes, Tenericutes, Proteobacteria, Verrucomicrobia, Euroarchaeota, for example, a bacterial taxon described in Table 2. In some embodiments, the human intestinal microbe It is a species with the bacterial taxa Actinobacteria. In some embodiments, the human gut microbe is a species with the bacterial taxa Bacteroidetes. In some embodiments, the human gut microbe is a species with the Firmicutes bacterial taxa. In some embodiments, the human gut microbe is a species with the bacterial taxa Fusobacteria. In some embodiments, the human gut microbe is a species with the Spirochaetes bacterial taxa. In some embodiments, the human gut microbe is a species with the bacterial taxa Synergistetes. In some embodiments, the human gut microbe is a species with the Tenericutes bacterial taxa. In some embodiments, the human gut microbe is a species with the bacterial taxa Proteobacteria. In some embodiments, the human gut microbe is a species with the Verrucomicrobia bacterial taxa. In some embodiments, the human gut microbe is a species with the Euroarchaeota bacterial taxa. In some embodiments, the human intestinal microbe is other than a Bifidobacterium or a Lactobacillus. In some embodiments, the glycan polymer (e.g., produced by a method described herein) is a substrate for a glycosidase enzyme from a human intestinal microbe of a certain CAZy family (e.g., a glycosylhydrolase (GH) family). or a glycosyltransferase (GT) family). In some embodiments, the glycan polymer is a substrate for a glycosidase enzyme from a human intestinal microbe of a particular glycosylhydrolase (GH) family (e.g., one from GH1 to GH135) or glycosyltransferase (GT) family (e.g., one from GT1 to GT101). In some embodiments, glycan polymer preparations are selected to be substrates for a human intestinal microbe with a particular glycosidase profile (e.g., expresses (or harbors in its genome) one or more glycosidase genes, e.g. one or more CAZy families). In some embodiments, glycosidase enzyme molecules are used in the methods described herein to produce glycan polymers that comprise functions of one or more of the glycosidase enzymes present in the particular microbe (or group of microbes). In some embodiments, the glycosidase enzyme molecule comprises the same functions as the glycosidase enzyme (e.g., the enzyme present in the intestinal microbe). In some embodiments, the glycosidase enzyme molecule has structural similarity or a certain degree of sequence similarity to the glycosidase enzyme. A glycosidase enzyme molecule can be generated by any method known in the art, for example, using conventional cloning, genetics, protein expression, protein purification or protein processing techniques.

Glycosidase enzyme molecules suitable for the glycan polymer preparation methods described herein can be selected based on their glycosidase enzyme homologues that are present in a microbe and, therefore, the glycan polymer or preparation thereof can adapt to the glycosidase enzyme (glycosidase enzyme profile) of the microbe as adapted substrates.

In some embodiments, the glycan polymer (e.g., produced by a method described herein) is a substrate for a glycosidase enzyme from a human intestinal microbe selected from the CAZy families GT5, GH94, GH13.9, GH13.39 , GH13.36, GH113.0 and GH112. In some embodiments, the glycan polymer is a substrate for a glycosidase enzyme from a human intestinal microbe selected from the CAZy families GT2, GT4, GT5, GT35, GT51, GH1, GH2, GH3, GH4, GH13, GH13 subfamily 9, subfamily 31 of GH13, GH18, GH23, GH25, GH28, GH31, GH32, GH36, GH51, GH73, GH77 and GH94. In some embodiments, the glycan polymer is a substrate for a glycosidase enzyme from a human intestinal microbe selected from the CAZy families GT11, GT10, GH92, GH51, GH35, GH29, GH28, GH20, GH130, GH13 subfamily 8 and subfamily 14 from GH13. In some embodiments, the glycan polymer is a substrate for a glycosidase enzyme from a human intestinal microbe selected from the CAZy families GT2, GT4, GH2, GH23, GH3, GT8, GT51, GT9, GH1, GH92, GH73, GH31, GH20 , GH28, GT25, GT28, GT35, GH18, GH13, GH36, GH97, GH105, GH25, GH4, GH32, GH78, GH29, GH51, GT10 and GH77. In some embodiments, the glycan polymer is a substrate for a glycosidase enzyme from a human intestinal microbe selected from the CAZy families GT3, GH97, GH43 subfamily 24, GH27, GH133, GH13 subfamily 8, and GH13. In some embodiments, the glycan polymer is a substrate for a glycosidase enzyme from a human intestinal microbe selected from the CAZy families GT2, GT4, GH2, GH23, GH3, GT51, GH1, GT8, GH92, GT9, GH73, GH31, GH20 , GH28, GT35, GT28, GH18, GH13, GH97, GH25, GH36, GH4, GH105, GH32, GH78, GH29, GT25, GH51, GH77, GH88, GH24.

In some embodiments, the glycosidase enzyme or glycosidase enzyme molecule is other than one of the CAZy family GH1, GH2, GH3, GH4, GH5, GH8, GH9, GH10, GH11, GH12, GH13, GH14, GH16, GH26, GH28, GH30, GH31, GH32, GH35, GH42, GH43, GH44, GH50, GH51, GH57, GH62, GH63, GH68, GH70, GH97, GH100, GH116, GH119 or GH122.

In some embodiments, the method described herein further comprises identifying a glycosidase profile (e.g., from the CAZy family (e.g., a GT family or a GH family)) of a particular microbe in silico. , the identification of a glycosidase profile is carried out according to the methods of examples 11-15. For example, the ability to modulate a metabolite from a sequenced genome can be predicted from a series of commensal bacterial species isolated from healthy human gut microbiomes, for example, as part of the Human Microbiome Project, e.g., produce butyrate, convert urea to ammonia via urease, or convert choline to TMA.

In some embodiments, the glycan polymer is a substrate for a glycosidase enzyme present in a microbe (e.g., a human gut microbe) that modulates the level of (e.g., produces) a microbial metabolite. Exemplary metabolites include formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, ascorbic acid, lactic acid, tryptophan, serotonin, indole, succinic acid, trimethylamine (TMA), TMAO ( trimethylamine N-oxide), deoxycholic acid, ethylphenyl sulfate, acetylaldehyde, hydrogen peroxide, ammonia, bile acids, lipopolysaccharide (LPS) and/or butanedione. In some embodiments, the metabolite is butyric acid (e.g., butyrate), trimethylamine (TMA), or ammonia. In some embodiments, the metabolite is butyric acid (e.g., butyrate). In some embodiments, the metabolite is acetic acid (e.g., acetate). In some embodiments, the metabolite is propionic acid (e.g., propionate). In some embodiments, the metabolite is trimethylamine (TMA). In some embodiments, the metabolite is ammonia. In some embodiments, the metabolite is lipopolysaccharide (LPS). In some embodiments, the metabolite is bile acid (e.g., a secondary bile acid). In some embodiments, a substantial increase or decrease in a metabolite may be detected. In some embodiments, the glycosidase enzyme or glycosidase enzyme molecule is other than alpha or beta-galactosidase, alpha or beta-glucosidase, alpha or beta-xylosidase, alpha or beta-mannosidase, or alpha or betafructofuranosidase. In some embodiments, the glycosidase enzyme or glycosidase enzyme molecule is other than alpha or beta-galactosidase.

Methods for generating glycosidase enzyme molecules

Glycosidase enzyme molecules can be produced by expression in recombinant host cells, but also by other methods such as in vitro transcription and translation and chemical synthesis.

For cellular expression, one or more nucleic acids (e.g., cDNA or genomic DNA) encoding a glycosidase enzyme molecule can be inserted into a replicable vector for cloning or expression. The vector may be, for example, a plasmid, cosmid, viral genome, phagemid, phage genome or other autonomously replicating sequence. The appropriate coding nucleic acid sequence can be inserted into the vector by a variety of methods. For example, they can engineer appropriate restriction endonuclease sites (e.g., using PCR). Restriction digestion and ligation can then be used to insert the coding nucleic acid sequence into an appropriate location. Vector components generally include one or more of an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

The glycosidase enzyme molecule may optionally be produced recombinantly by fusion with one or more different components, such as a signal sequence, an epitope or purification moiety or a marker.

For bacterial expression, the glycosidase enzyme molecule can be produced with or without a signal sequence. For example, it can be produced within cells so that it accumulates in inclusion bodies or in the soluble fraction. It can also be secreted, for example, by the addition of a prokaryotic signal sequence, for example, an appropriate leader sequence. Exemplary bacterial host cells for expression include any transformable strain of E. coliK-12 (such as E. coliBL21, C600, ATCC 23724; E. coliHB101 NRRLB-11371, ATCC-33694; E. coliMM294 ATCC-33625; E. coliW3110 ATCC-27325), strains ofB. subtilis, Pseudomonas and other bacilli. In some embodiments, the bacterial host cell is selected from a proteolytic taxon, for example, a taxon that expresses few or no endogenous glycosidase enzymes.

Glycosidase enzyme molecules can be expressed in a yeast host cell, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Hanseula or Pichia pastoris. For expression in yeast, glycosidase enzyme molecules can also be produced intracellularly or by secretion, for example using the yeast invertase leader or an alpha factor leader (including Saccharomyces and Kluyveromyces forms), or the acid phosphatase leader, or the glucoamylase leader of C. albicans (document EP 362.179 published on April 4, 1990).

Both expression and cloning vectors contain a nucleic acid sequence that allows the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeasts and viruses. The plasmid pBR322 origin of replication is suitable for most Gram-negative bacteria; the origin of plasmid 2^ is suitable for yeast.

Expression and cloning vectors usually contain a gene or selection marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, for example, ampicillin, neomycin, methotrexate or tetracycline, (b) complement auxotrophic deficiencies (such as the URA3 marker in Saccharomyces), or (c) supply critical nutrients not available from complex media, for example, the gene encoding D-alanine racemase for bacilli.

Expression and cloning vectors typically contain a promoter operatively linked to the nucleic acid sequence encoding the glycosidase enzyme molecule to direct mRNA synthesis. Exemplary promoters suitable for use with prokaryotic hosts include the p-lactamase and lactose promoter systems (Changet al., Nature, 275:615 (1978); Goeddelet al., Nature, 281:544 (1979)). , alkaline phosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776) and hybrid promoters such as the tac promoter (deBoeret al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983). Promoters for use in bacterial systems may also contain an appropriately located Shine-Dalgarno sequence. The T7 polymerase system can also be used to direct the expression of a nucleic acid coding sequence placed under the control of the T7 promoter. Still other methods, vectors and host cells suitable for adaptation to the synthesis of glycosidase enzyme molecules in recombinant cells are described in Molecular Cloning: A Laboratory, third edition, Sambrooket al. (eds.), Cold Spring Harbor Press, (2001 ) (ISBN: 0879695773).

Once expressed in cells, glycosidase enzyme molecules can be recovered from culture medium, inclusion bodies, or cell lysates. Cells can be disrupted by various physical or chemical means, such as freeze-thaw cycles, sonication, mechanical disruption, or cell lysis agents (e.g., detergents).

Glycosidase enzyme molecules can be purified from other cellular proteins or polypeptides that can be found in cell lysates or in the cellular medium. Various protein purification methods can be employed and such methods are known in the art and are described, for example, in Deutscher, Methods in Enzymology, 182 (1990); and Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (2010) (ISBN: 1441928332). Examples of purification procedures include: by fractionation on an ion exchange column; ethanol precipitation; Reversed phase HPLC; chromatography on silica or on a cation exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG; and affinity columns (for example, metal chelating columns to bind epitope-tagged forms of the protein and columns with various ligands to bind any purification moieties that are associated with the glycosidase enzyme). A purification method may include a combination of two different ion exchange chromatography steps, for example, cation exchange chromatography followed by anion exchange chromatography, or vice versa. The glycosidase enzyme molecules can be eluted from the ion exchange resin by a variety of methods including salt and/or pH gradients or steps. In some embodiments, the glycosidase enzyme molecules include a purification moiety (such as epitope tags and affinity identifiers). Such residues can be used for affinity chromatography and can optionally be removed by proteolytic cleavage.

Anionic or cationic substituents can be attached to matrices in order to form anionic or cationic supports for chromatography. Anion exchange substituents include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), and quaternary amine (Q) groups. Cationic substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P), and sulfonate (S). Cellulose ion exchange resins such as De23, DE32, DE52, CM-23, c M-32 and CM-52 are available from Whatman Ltd. (Maidstone, Kent, UK). SEFADEX™ and other cross-linked ion exchangers are also known. For example, DEAE-, QAE-, CM- and SP-SEPHADEX™ and DEAE-, Q-, CM- and S-SEPHAROSE™ and SEPHAROSE™ Fast Flow are available from Pharmacia AB. Ethylene glycol methacrylate copolymers derivatized with DEAE and CM, such as TOYOPEARL DEAE-650S or M and TOYOPEARL CM-650S or M, are available from Toso Haas Co. (Philadelphia, PA, USA).

A cation exchange surface is an ion exchange surface with negatively charged ligands covalently attached and therefore has free cations for exchange with cations in a solution in contact with the surface. Exemplary surfaces include cation exchange resins, such as those wherein the covalently attached groups are carboxylate or sulfonate. Commercially available cation exchange resins include CMC-cellulose, SP-Sephadex™ and Fast S-Sepharose™ (Pharmacy).

An anion exchange surface is an ion exchange surface with covalently attached positively charged groups, such as quaternary amino groups. An exemplary anion exchange surface is an anion exchange resin, such as DEAE cellulose, TMAE, QAE Sephadex™ and Fast Q Sepharose™ (Pharmacy).

An exemplary purification scheme for glycosidase enzyme molecules includes lysing E cells. colin lysis buffer followed by depth filtration. The material is then subjected to cation exchange chromatography (CEX). The CEX eluate is then flowed over an anion exchange medium in an anion exchange chromatography (AEX) step. The AEX FT can undergo a polishing stage. The material can then be processed by ultrafiltration/diafiltration, for example, to concentrate or desalt the material. Ultrafiltration/diafiltration membranes can be selected based on nominal molecular weight cutoff (“NMWCO”) to retain protein in the retentate, while allowing low molecular weight materials such as salts to pass into the filtrate. Any buffer solution or sterile water can be used during the final buffer exchange step, for example, depending on the desired final pH and conductivity of the product.

A glycosidase enzyme molecule may comprise one or more conservative sequence modifications. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced by conventional techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which the amino acid residue is replaced with an amino acid residue that has a similar side chain. Families of amino acid residues that have similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), polar uncharged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains ( for example, tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues within a glycosidase enzyme can be replaced with other amino acid residues from the same side chain family and the altered glycosidase enzyme molecule can be tested using the functional assays described herein.

Additional processing steps

The following aspects and embodiments related to additional processing steps are for illustrative purposes.

Optionally, the preparation may undergo additional processing steps. Additional processing steps may include, for example, purification steps. Purification steps may include, for example, separation, dilution, concentration, filtration, desalting or ion exchange, chromatographic separation or decolorization, or any combination thereof.

Discoloration

In some embodiments, the methods described herein further include a bleaching step. A produced glycan polymer may be subjected to a decolorization step using any method known in the art, including, for example, treatment with an adsorbent, activated carbon, chromatography (e.g., using ion exchange resin), hydrogenation and/or filtration. (e.g. microfiltration).

In certain embodiments, a produced glycan polymer is contacted with a color absorbing material at a particular temperature, at a particular concentration and/or for a particular period of time.

In some embodiments, the mass of the color absorbing species in contact with a glycan polymer is less than 50% of the mass of the glycan polymer, less than 35% of the mass of the glycan polymer, less than 20% of the mass of the glycan polymer, less than 10% of the mass of the glycan polymer, less than 5% of the mass of the glycan polymer, less than 2% of the mass of the glycan polymer or less than 1% of the mass of the glycan polymer.

In some embodiments, a glycan polymer is contacted with a color absorbing material. In certain embodiments, a glycan polymer is contacted with a color absorbing material for less than 10 hours, less than 5 hours, less than 1 hour, or less than 30 minutes. In a particular embodiment, a glycan polymer is contacted with a color absorbing material for 1 hour. In certain embodiments, the glycan polymer is contacted with a color absorbing material at a temperature of 20 to 100 degrees Celsius, 30 to 80 degrees Celsius, 40 to 80 degrees Celsius or 40 to 65 degrees Celsius. In a particular embodiment, the glycan polymer is contacted with a color absorbing material at a temperature of 50 degrees Celsius.

In certain embodiments, the color absorbing material is activated carbon. In one embodiment, the color absorbing material is powdered activated carbon. In other embodiments, the color absorbing material is an ion exchange resin. In one embodiment, the color absorbing material is a strong base cation exchange resin in chloride form. In another embodiment, the color absorbing material is cross-linked polystyrene. In yet another embodiment, the color absorbing material is cross-linked polyacrylate. In certain embodiments, the color absorbent material is Amberlite FPA91, Amberlite FPA98, Dowex 22, Dowex Marathon MSA or Dowex Optipore SD-2.

Ion exchange/desalting (demineralization)

In some embodiments, the produced glycan polymer is contacted with a material to remove salts, minerals and/or other ionic species. In certain embodiments, the glycan polymer is flowed through a pair of anion/cation exchange columns. In one embodiment, the anion exchange column contains a weak base exchange resin in hydroxide form and the cation exchange column contains a strong acid exchange resin in protonated form.

Separation and concentration

In some embodiments, the methods described herein further include isolating the produced glycan polymers. In certain variations, isolating glycan polymers comprises separating at least a portion of the glycan polymers from at least a portion of the glycosidase enzyme molecule, using any method known in the art, including, for example, centrifugation, filtration (e.g. , vacuum filtration, membrane filtration) and gravity sedimentation. In some embodiments, isolating the glycan polymers comprises separating at least a portion of the glycan polymers from at least a portion of any unreacted sugar, using any method known in the art, including, for example, filtration (e.g., filtration by membrane), chromatography (for example, chromatographic fractionation), differential solubility and centrifugation (for example, differential centrifugation).

In some embodiments, the methods described herein further include a concentration step. For example, in some embodiments, the isolated glycan polymer is subjected to evaporation (e.g., vacuum evaporation) to produce a concentrated glycan polymer preparation. In other embodiments, the isolated glycan polymer is subjected to a spray drying step to produce an oligosaccharide powder. In certain embodiments, the isolated glycan polymer is subjected to both an evaporation step and a spray drying step.

Division

In some embodiments, the methods described herein further include a fractionation step. The prepared and purified glycan polymers can subsequently be separated by molecular weight using any method known in the art, including, for example, high-performance liquid chromatography, adsorption/desorption (e.g., low-pressure activated carbon chromatography), or filtration. (e.g. ultrafiltration or diafiltration).

In certain embodiments, the produced glycan polymers are fractionated by adsorption onto a carbonaceous material and subsequent desorption of fractions by washing the material with mixtures of an organic solvent in water at a concentration of 1%, 5%, 10%, 20%. %or 100%. In one embodiment, the adsorption material is activated carbon. In another embodiment, the adsorption material is a mixture of activated carbon and a bulking agent such as diatomaceous earth or Celite 545 in a portion of 5%, 10%, 20%, 30%, 40% or 50% by volume. or weight.

In additional embodiments, the glycan polymers produced are separated by passage through a high-performance liquid chromatography system. In certain variations, the glycan polymers produced are separated by ion affinity chromatography, hydrophilic interaction chromatography, or size exclusion chromatography, including gel permeation and gel filtration.

In other embodiments, low molecular weight materials are removed by filtration methods. In certain variations, low molecular weight materials can be removed by dialysis, ultrafiltration, diafiltration, or tangential flow filtration. In certain embodiments, filtration is performed in a static dialysis tubing apparatus. In other embodiments, filtration is performed in a dynamic flow filtration system. In other embodiments, filtration is performed in filtration cartridges driven by centrifugal force.

Other processing steps may include any of those described in the examples herein. In some embodiments, yeast fermentation is used to remove unreacted constituents, for example, sugar monomers or dimers, or reaction byproducts, such as sugar monomers.

Properties of the glycan preparation

The glycan may have any one or more of the characteristics and properties disclosed in WO2016/122889, WO2016/172657, WO 2016/007778 and WO2016/172658, and any characteristics and properties disclosed herein.

Glycans produced by the methods described herein may comprise oligosaccharides. In some embodiments, the glycans comprise homo-oligosaccharides (or homoglycans), where all of the monosaccharides in a polymer are of the same type.

In some embodiments, the glycans comprise hetero-oligosaccharides (or heteroglycans), where more than one type of monosaccharide is present in the polymer. In some embodiments, the glycans have one or more of the properties described herein. In some embodiments, the glycan preparation has one or more of the bulk properties described herein.

Degree of polymerization (DP)

In some embodiments, glycan polymer preparations are produced, for example, using a method described herein, that are polydisperse, exhibiting a range of degrees of polymerization. Optionally, the preparations can be fractionated, representing for example 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or more than 98% of short species (approximately DP1-2), medium (approximately DP3-10), long (approximately DP11-18) or very long (approximately DP>18). In one embodiment, a fractionated, polydisperse glycan polymer preparation is provided comprising at least 85%, 90%, or at least 95% mid-length species with a DP of about 3-10. In one embodiment, a fractionated, polydisperse glycan polymer preparation is provided comprising at least 85%, 90%, or at least 95% long-length species with a DP of about 11-18. In one embodiment, a fractionated, polydisperse glycan polymer preparation is provided comprising at least 85%, 90%, or at least 95% very long length species with a DP of about 18-30.

Optionally, the preparations can be fractionated, representing for example 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or more than 98% short glycans (approximately DP1-2) or media (approximately DP3-10) in the preparation. Alternatively, or in addition to fractionation, the small DP fraction (e.g. monomers and dimers) is subjected to enzymatic fermentation, e.g. with yeasts suitable for breaking down these sugars. In one embodiment, a polydisperse and fractionated glycan polymer preparation is prepared using a method described herein, comprising at least 85%, 90%, or at least 95% glycans with a DP of about 3- 10. In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymers of the glycan preparation have a DP of at least DP3, DP4, DP5, DP6 or DP7. In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymers of the glycan preparation have a DP from about DP3 to about DP10, from about DP3 to about DP8, from about DP3 to about DP6, from about DP3 to about DP5, from about DP3 to about DP4, from about DP2 to about DP4, from about DP2 to about DP5, from about DP2 to about DP6, from about DP2 to about DP8 or from about DP2 to about Dp10. In some embodiments, less than 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 40% or less than 50% of the glycan polymers of the glycan preparation have a DP of DP2 or less.

In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymer preparation has a DP of between 2 and 25, between 3 and 25, between 4 and 25, between 5 and 25, between 6 and 25, between 7 and 25, between 8 and 25, between 9 and 25, between 10 and 25, between 2 and 30, between 3 and 30, between 4 and 30, between 5 and 30, between 6 and 30, between 7 and 30, between 8 and 30, between 9 and 30 or between 10 and 30. In one embodiment, the glycan polymer preparation has a degree of polymerization (DP) of at least 3 and less than 30 glycan units.

In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymer preparation has a DP of at least 5 and less than 30 glycan units. In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymer preparation has a DP of at least 8 and less than 30 glycan units. In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymer preparation has a DP of at least 10 and less than 30 glycan units. In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymer preparation has a DP of between 3 , 4, 5, 6, 7, 8 and 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 glycan units. In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymer preparation has a DP of between 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 glycan units. In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymer preparation has a DP of between 3 , 4, 5, 6, 7, 8, 9, 10 and 20, 21, 22, 23, 24, 25, 26, 27, 28 glycan units.

The conversion yield for the one or more glycan units (e.g., sugars) in the methods described herein can be determined by any suitable method known in the art, including, for example, high-performance liquid chromatography (HPLC). ). The average conversion yield can be determined by methods known to those skilled in the art, for example size exclusion, ionic affinity, hydrophilic or hydrophobic chemistry. These methods are generally based on the chromatographic separation of materials using an HPLC system equipped with appropriate column chemistry. Chromatographic separation of starting materials from products then allows direct comparison of the area under the curve of those materials which can then be converted into a percentage conversion yield. Example 15 describes specific IAC and SEC approaches that can be used to determine conversion performance. In a preferred embodiment, the conversion as mentioned herein is determined by the SEC method of Example 15.

In some embodiments, the conversion yield for a glycan polymer preparation with glycan polymers of a DP of more than DP1 (DP>1) after combining the one or more glycan subunits with the glycosidase enzyme molecule is greater than or equal to approximately 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% 60%, 65%, 70%, 75% (as determined on a w/w basis as a percentage of the input glycan subunits). In some embodiments, the conversion yield for a glycan polymer preparation with glycan polymers of a DP of at least DP2 after combining the one or more glycan subunits with the glycosidase enzyme molecule is greater than or equal to about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% , 70%, 75% (as determined on a weight/weight basis as a percentage of the input glycan subunits).

In some embodiments, the conversion yield for a glycan polymer preparation with glycan polymers of a DP of at least DP3 after combining the one or more glycan subunits with the glycosidase enzyme molecule is greater than or equal to about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% , 70%, 75% (as determined on a weight/weight basis as a percentage of the input glycan subunits).

In some embodiments, the conversion yield for a glycan polymer preparation with DP > 1 after combining the one or more glycan units with the catalyst (e.g., at 2, 3, 4, 8, 12, 24 or 48 hours after combining the one or more glycan units with the catalyst) is greater than about 50% (e.g., greater than about 55%, 60%, 65%, 70%, 75%, 80%, 85 %, 90%, 95% or 98%). In some embodiments, the conversion yield for a glycan polymer preparation with > DP2 after combining the one or more glycan units with the catalyst (e.g., at 2, 3, 4, 8, 12, 24, or 48 hours after combining the one or more glycan units with the catalyst) is greater than 30% (e.g., greater than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%).

In one embodiment, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymer preparation has a DP of at least 2. In one embodiment, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymer preparation has a DP of at least 3.

average DP

In some embodiments, the glycan polymer preparation has an average degree of polymerization (average DP) of about DP2, DP3, DP4, DP5, DP6, DP7, DP8 or DP9. In some embodiments, the glycan polymer preparation has an average degree of polymerization (average DP) of between about 2 and about 10, between about 2 and about 8, between about 2 and about 6, between about 2 and about 4, between about 3 and about 10, between about 3 and about 8, between about 3 and about 6, or between about 3 and about 4.

In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymer preparation has an average degree of polymerization. (Dp) of approximately DP5, DP6, DP7, Dp8, DP9, DP10, DP11 or DP12. In some embodiments, the average DP of the glycan polymer preparation is between about DP5 and DP10, between about Dp6 and DP10, between about DP6 and DP12, between about DP6 and DP14, between about DP8 and DP12, between about DP8 and DP14, between approximately DP8 and DP16, between approximately DP10 and DP16, between approximately DP10 and DP18, between approximately DP4 and DP18, between approximately DP6 and DP18 or between approximately DP8 and DP18.

The (or average) degree of polymerization (DP) distribution of a glycan polymer preparation can be determined by methods known to those skilled in the art, for example using ion affinity chromatography (IAC) or size exclusion measurements ( SEC) of the molecular weight (MW) followed by a mathematical conversion into average DP. These methods are generally based on the chromatographic separation of materials based on an HPLC system equipped with mass-sensitive column chemistry such as size exclusion or ion affinity columns followed by a computational conversion of that distribution into an average MW. by comparison with a set of patterns with known PM. Once the average MW is determined, dividing that value by the average weight of the glycan repeat unit allows calculation of the average DP. Example 15 describes specific IAC and SEC approaches that can be used to determine the average DP as mentioned herein. In a preferred embodiment, the average DP as mentioned herein is determined by the SEC method of Example 15.

average molecular weight

In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymers of the preparation have an average molecular weight of approximately 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, , 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800 g/mol and less than 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 0, 1600, 1700 , 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200 , 4300, 4400, 4500, 4600, 4700, 4800, 4900 and 5000 g/mol.

The average molecular weight (MW) can be determined by methods known to those skilled in the art, for example, ion affinity chromatography (IAC) or size exclusion chromatography (SEC). These methods are generally based on the chromatographic separation of materials based on an HPLC system equipped with mass-sensitive column chemistry such as size exclusion or ion affinity columns followed by a computational conversion of that distribution into an average MW. by comparison with a set of patterns with known PM. Example 15 describes specific IAC and SEC approaches that can be used to determine the average PM as mentioned herein. In a preferred embodiment, the average MW as mentioned herein is determined by the SEC method of Example 15.

Degree of branching (DB)

In some embodiments, the structure of the glycan preparations ranges from linear to branched. Branched glycans may contain at least one glycan subunit that is linked via an alpha or beta glycosidic bond to form a branch. The branching rate or degree of branching (DB) may vary, such that the glycan polymers of a preparation comprise at least 1, at least 2, at least 3, at least 4, at least 5 or at least about 6 points branching in the glycan polymer. In some embodiments, the glycan polymers of the glycan preparation are unbranched (DB=0).

In some embodiments, the structure of the glycan preparations (e.g., oligo- or polysaccharides) ranges from linear to highly branched. Unbranched glycans may contain only alpha linkages or only beta linkages. Unbranched glycans may contain at least one alpha and at least one beta linkage. Branched glycans may contain at least one glycan unit that is linked via an alpha or a beta glycosidic bond to form a branch. The branching rate or degree of branching (DB) can vary, so that approximately every 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 15th, 20th, 25th, 30th, 35th, 40th, 45th , 50th, 60th or 70th unit comprises at least one branch point. For example, animal glycogen contains a branch point approximately every 10 units.

In some embodiments, glycan polymer preparations are provided wherein the preparation comprises a mixture of branched glycans, wherein the average degree of branching (DB, branch points per residue) is 0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0, 6, 0.7, 0.8, 0.9, 0.95, 0.99, 1 or 2. In some embodiments, glycan polymer preparations are provided where the average degree of branching is at least 0, 01, 0.05, 0.1, 0.2, 0.3 or at least 0.4. In some embodiments, glycan polymer preparations are provided wherein the average degree of branching is between about 0.01 and 0.1, 0.01 and 0.2, 0.01 and 0.3, 0.01 and 0.4, 0.01 and 0.5, 0.01 and 0.6, or between approximately 0.01 and 0.7. In some embodiments, glycan polymer preparations are provided wherein the average degree of branching is between about 0.05 and 0.1, 0.05 and 0.2, 0.05 and 0.3, 0.05 and 0.4, 0.05 and 0.5, 0.05 and 0.6, or between approximately 0.05 and 0.7. In some embodiments, glycan polymer preparations are provided where the average degree of branching is not 0. In some embodiments, glycan polymer preparations are provided where the average degree of branching is not between at least 0.1 and minus 0.4 or at least 0.2 and minus 0.4. In some embodiments, the glycan polymer preparations comprise linear glycans. In some embodiments, glycan polymer preparations comprise glycans that exhibit a branched or branched-branched structure.

In some embodiments, glycan polymer preparations are provided wherein the average degree of branching (DB) is not 0, but is at least 0.01, 0.05, 0.1 or at least 0.2, or ranges from about 0.01 to about 0.2 or from about 0.05 to 0.1.

The degree of branching (DB) of a glycan polymer preparation can be determined by methods known to those skilled in the art, for example, permethylation analysis. These methods are generally based on chemical functionalization of free hydroxyl groups of a glycan followed by total acid hydrolysis and GC-MS analysis of the isolated monomers. Therefore, the fraction of monomers with multiple non-functionalized hydroxyl groups can be interpreted to be equal to the fraction of polymer units that were linked to more than one other unit, for example, the branched fraction. Example 15 describes specific permethylation approaches that can be used to determine the DB as mentioned herein. In a preferred embodiment, the DB as mentioned herein is determined by permethylation of example 15.

Glycosidic bonds and bonds

Linkages between individual glycan subunits found in glycan polymer preparations may include alpha 1->2, alpha 1->3, alpha 1->4, alpha 1->5, alpha 1->6, alpha 2->1, alpha 2->3, alpha 2->4, alpha 2->6, beta 1->2, beta 1->3, beta 1->4, beta 1->5, beta 1- >6, beta 2->1, beta 2->3, beta 2->4 and beta 2->6.

In some embodiments, the glycan polymer preparations comprise only alpha linkages. In some embodiments, the glycan polymers comprise only beta linkages. In some embodiments, the glycan polymers comprise mixtures of alpha and beta linkages.

In some embodiments, the alpha:beta glycosidic bond ratio in a preparation is about 1:1, 2:1, 3:1, 4:1, or 5:1. In some embodiments, the beta:alpha glycosidic bond ratio in a preparation is about 1:1, 2:1, 3:1, 4:1, or 5:1.

In some embodiments, the alpha:beta glycosidic bond ratio in a preparation is about 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0. 6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.2:1, 1.5:1, 1.7:1, 2:1, 2, 2:1, 2.5:1, 2.7:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or approximately 10:1.

In some embodiments, the glycan polymers of the glycan polymer preparation comprise both alpha and beta glycosidic linkages selected from the group consisting of 1->2 glycosidic linkage, a 1->3 glycosidic linkage, a 1->4 glycosidic linkage. , a 1->5 glycosidic bond and a 1->6 glycosidic bond. In some embodiments, the glycan polymer preparation comprises at least two or at least three alpha and beta 1->2 glycosidic bonds, alpha and beta 1->3 glycosidic bonds, alpha and beta 1->4 glycosidic bonds, alpha and beta 1->5 and/or alpha and beta 1->6 glycosidic bonds.

In some embodiments, the glycan polymers of the glycan preparation comprise substantially all of the alpha- or beta-configured glycan subunits, optionally comprising about 1%, 2%, 3%, 4% 5%, 6%, 7%. 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% of the respective other configuration.

In some embodiments, the glycan polymer preparations comprise at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%. 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% , 99%, at least 99.9% or even 100% glycans with alpha glycosidic bonds. In some embodiments, the glycan polymer preparations comprise at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%. 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% , 99%, at least 99.9% or even 100% glycans with beta glycosidic bonds. In some embodiments, glycan polymer preparations are provided wherein at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% % 70%, 75%, 80% or at least 85% glycans with glycosidic linkages that are alpha glycosidic linkages, at least 10%, 15%, 20%, 25%, 30%, 35%, 40% , 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or at least 85% of glycans with glycosidic bonds that are beta glycosidic bonds, and wherein the percentage of alpha glycosidic bonds and beta does not exceed 100%.

In some embodiments, glycan polymer preparations are provided wherein at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% %, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% 98%, 99%, at least 99.9% or even 100% of the glycosidic bonds in the glycan are one or more of: glycosidic bonds 1->2, glycosidic bonds 1->3, glycosidic bonds 1->4 and glycosidic bonds 1->6. In some embodiments, glycan polymer preparations are provided wherein at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, at At least 20% or 25% of each of the glycosidic bonds in the glycan are 1->2, 1->3, 1->4 and 1->6 glycosidic bonds. Optionally, the glycan polymer preparations further comprise at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% %, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or at least 85% glycosidic linkages of the glycan that are selected from the group consisting of: glycosidic bonds alpha 2->1, alpha 2->3, alpha 2->4, alpha 2->6, beta 2->1, beta 2->3, beta 2->4 and beta 2 - >6.

In some embodiments, the glycan polymers of the glycan preparation comprise at least two glycosidic linkages selected from the group consisting of alpha 1->2 and alpha 1->3, alpha 1->2 and alpha 1->4, alpha 1->2 and alpha 1->6, alpha 1->2 and beta 1->2, alpha 1->2 and beta 1->3, alpha 1->2 and beta 1->4, alpha 1- >2 and beta 1->6, alpha 1->3 and alpha 1->4, alpha 1->3 and alpha 1->6, alpha 1->3 and beta 1->2, alpha 1->3 and beta 1->3, alpha 1->3 and beta 1->4, alpha 1->3 and beta 1->6, alpha 1->4 and alpha 1->6, alpha 1->4 and beta 1->2, alpha 1->4 and beta 1->3, alpha 1->4 and beta 1->4, alpha 1->4 and beta 1->6, alpha 1->6 and beta 1- >2, alpha 1->6 and beta 1->3, alpha 1->6 and beta 1->4, alpha 1->6 and beta 1->6, beta 1->2 and beta 1->3 , beta 1->2 and beta 1->4, beta 1->2 and beta 1->6, beta 1->3 and beta 1->4, beta 1->3 and beta 1->6, and beta 1->4 and beta 1->6.

The distribution of glycosidic linkages and bonds can be determined by methods known to those skilled in the art, for example, two-dimensional nuclear magnetic resonance spectroscopy (2D NMR). These methods are generally based on quantifications of the area under the curve (AUC) of diagnostic peaks for a given junction type. Example 15 describes specific 2D NMR approaches that can be used to determine glycosidic linkages and bonds as mentioned herein. In a preferred embodiment, glycosidic linkages and bonds are determined using the 2D NMR method of Example 15.

Forms L and D

In some embodiments, glycan polymer preparations are provided where at least one glycan subunit is an L-form sugar. In some embodiments, glycan preparations are provided where at least one glycan subunit is a D-form sugar. In some embodiments, glycan preparations are provided wherein the glycan subunits are L- or D-form sugars as they occur naturally or are more common (e.g., D-glucose, D-xylose, L-arabinose).

In some embodiments, the preparation of glycan polymers (e.g., oligosaccharides and polysaccharides) comprises a desired mixture of L and D forms of glycan subunits, e.g., of a desired ratio, such as: 1:1, 1:2 , 1:3, 1:4, 1:5 of L-forms with respect to L or of D-forms with respect to L.

In some embodiments, the preparation of glycan polymers comprises a desired mixture of L and D forms of glycan units, for example of a desired ratio, such as: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:14, 1:16, 1:18, 1:20, 1:25, 1: 30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:100, 1:150 of L shapes with respect to D or of D shapes with respect to L.

In some embodiments, the glycan polymer preparation comprises glycans with substantially all L or D forms of glycan subunits, optionally comprising about 1%, 2%, 3%, 4% 5%, 6%, 7%. 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% in the other respective way.

Glycan unit content

In some embodiments, glycan polymer preparations are provided where at least one glycan subunit is a tetrose, a pentose, a hexose or a heptose. Optionally, the glycan subunits involved in the formation of the glycans of the glycan polymer preparation vary. Examples of monosaccharide glycan subunits include hexoses, such as glucose, galactose and fructose, and pentoses, such as xylose. Monosaccharides generally have the chemical formula: Cx(H2O)y, where conventionally x > 3. Monosaccharides can be classified by the number x of carbon atoms they contain, for example: goddess (2) triose (3) tetrose (4) , pentose (5), hexose (6) and heptose (7). Monosaccharide glycan subunits can exist in an acyclic (open chain) form. Open-chain monosaccharides with the same molecular graph can exist as two or more stereoisomers. Monosaccharides can also exist in a cyclic form through a nucleophilic addition reaction between the carbonyl group and one of the hydroxyls of the same molecule. The reaction creates a ring of carbon atoms closed by a bridging oxygen atom. In these cyclic forms, the ring usually has 5 (furanoses) or 6 (pyranoses) atoms.

In some embodiments, the glycan polymer preparation comprises a desired mixture of different monosaccharide glycan subunits, such as a mixture of a goddess (2), a triose (3), tetrose (4), pentose (5), hexose (6) or heptose (7). In some embodiments, the glycan polymers of the glycan polymer preparation comprise a desired mixture of a pentose (5) and a hexose (6).

In some embodiments, the glycan polymer preparation comprises a desired mixture of two, three, four or five different glycan subunits, such as a mixture of, for example, i) one or more glycan subunits selected from monosaccharides, selected from glucose, a galactose, an arabinose, a mannose, a fructose, a xylose, a fucose and a rhamnose; ii) one or more glycan subunits selected from disaccharides selected from acarviosin, n-acetylactosamine, allolactose, cellobiose, chitobiose, galactose-alpha-1,3-galactose, gentiobiose, isomalt, isomaltose, isomaltulose, kojibiose, lactitol, lactobionic acid, lactose, lactulose, laminaribose, maltitol, maltose, mannobiose, melibiose, melibiulose, neohesperidose, nigerose, robinose, rutinose, sambubiose, sophorose, sucralose, sucrose, sucrose acetate isobutyrate, sucrose octaacetate, trehalose, turanose, vicianose and xylobiose; iii) one or more glycan subunits selected from amino sugars selected from acarbose, N-acetylmannosamine, N-acetylmuramic acid, N-acetylneuraminic acid, N-acetylthalosaminuronic acid, arabinopyranosyl-N-methyl-N-nitrosourea, D-fructose-L- histidine, N-glycolineuraminic acid, ketosamine, kidamycin, mannosamine, 1B-methylselene-N-acetyl-D-galactosamine, muramic acid, muramyl dipeptide, phosphoribosylamine, PUGNAc, sialyl-Lewis A, sialyl-Lewis X, validamycin, voglibose, N -acetylgalactosamine, N-acetylglucosamine, aspartylglucosamine, bacilthiol, daunosamine, desosamine, fructosamine, galactosamine, glucosamine, meglumine and perosamine; iv) one or more glycan subunits selected from deoxysugars selected from 1-5-ahydroglucitol, cladinose, colitose, 2-deoxy-D-glucose, 3-deoxyglucasone, deoxyribose, dideoxynucleotide, digitalose, fludeoxyglucose, sarmentose and sulfoquinovose; v) one or more glycan subunits selected from iminosugars selected from castanospermine, 1-deoxynojirimycin, iminosugar, miglitol, miglustat and swainsonine; one or more glycan subunits selected from sugar acids selected from N-acetylneuraminic acid, N-acetylthalosamnuronic acid, aldaric acid, aldonic acid, 3-deoxy-D-manno-oct-2-ulosonic acid, glucuronic acid, glucosaminouronic acid, glyceric acid, N-glycolylneuraminic acid, iduronic acid, isosacharinic acid, pangamic acid, sialic acid, threonic acid, ulosonic acid, uronic acid, xylonic acid, gluconic acid, ascorbic acid, ketodeoxyoctulosonic acid, galacturonic acid, galactosaminuronic acid, mannuronic acid, mannosaminuronic acid, tartaric acid, mucic acid, saccharic acid, lactic acid, oxalic acid, succinic acid, hexanoic acid, fumaric acid, maleic acid, butyric acid, citric acid, glucosamine acid, malic acid, succinamic acid, sebacic acid and capric acid ; vi) one or more glycan subunits selected from short chain fatty acids selected from formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid; and vii) one or more glycan subunits selected from sugar alcohols selected from methanol, ethylene glycol, glycerol, erythritol, threitol, arabitol, ribitol, xylitol, mannitol, sorbitol, galactitol, iditol, volemitol, fucitol, inositol, maltotritol, maltotetraitol and polyglycitol.

Exemplary glycans are described by a three-letter code representing the monomeric sugar component followed by a number in one hundred reflecting the percentage of the material that constitutes the monomer. Therefore, 'glu100' is attributed to a glycan generated from a 100% supply of D-glucose (glycan unit) and 'glu50gal50' is attributed to a glycan generated from a 50% supply of D -glucose and 50% of D-galactose (glycan units) or, alternatively, from a contribution of lactose dimer (glycan unit). As used herein: xyl = D-xylose; ara = L-arabinose; gal = D-galactose; glu = D-glucose; rha = L-rhamnose; fuc = L-fucose; man = D-mannose; sor = D-sorbitol; gly = D-glycerol; neu = NAc-neuraminic acid.

In some embodiments, the glycan polymer preparation comprises a glycan unit A selected from i) to vii) above, wherein the glycan unit A comprises 100% of the glycan unit contribution. For example, in some embodiments, the glycan polymer preparation is selected from the homoglycans xyl100, rha100, ara100, gal 100, glu100 and man100. In some embodiments, the glycan polymer preparation is selected from the fuc100 and fru100 homoglycans.

In some embodiments, the preparation of glycan polymers comprises a mixture of two glycan units A and B independently selected from i) to vii) above, where A and B may be selected from the same or different group i) to vii) and in where A and B can be selected at any desired ratio (for example, between 1-99% of A and between 99-1% of B, without exceeding 100%).

For example, in some embodiments, the glycan polymer preparation is selected from the heteroglycans ara50gal50, ara50gal50, xyl75gal25, ara80xyl20, ara60xyl40, ara50xyl50, glu80man20, glu60man40, man80glu20, man60glu40, gal20, gal75xyl25, Man66gal33, Man75gal25, glu80gal20, glu60gal40, glu40gal60, glu20gal80, gal80man20, gal60man40, gal40man60, glu80xyl20, glu60xyl40, glu40xyl60, glu20xyl80, glu80ara20, glu60ara40, glu40ara60, glu20ara80, gal8 0xyl20, gal 60xyl40, gal40xyl60, gal20xyl80, gal80ara20, gal60ara40, gal40ara60, gal20ara80, man80xyl20, man60xyl40 , man40xyl60, man20xyl80, man80ara20, man60ara40, man40ara60, man20ara80, xyl80ara20, xyl60ara40, glu50gal50 and man62glu38.

In some embodiments, the glycan polymer preparation comprises a mixture of three glycan units A, B and C independently selected from i) to vii) above, where A, B and C may be selected from the same or a different group i ) to vii) and where A, B and C can be selected at any desired ratio (for example, anywhere between 1-99% of A, 1-99% of B, 1-99% of C, without exceeding 100%).

For example, in some embodiments, the glycan polymer preparation is selected from the heteroglycans xyl75glu12gal12, xyl33glu33gal33, , man52glu29gal19, Glu33Man33Xyl33, Glu33Man33Ara33, Glu33Xyl33Ara33, Gal33Man33Xyl33, Gal33Man33Ara33, Gal33Xyl33Ara33, Man33Xyl33Ara33, Glu90Gal5Man5, Glu80Gal10Man10, Glu60Gal20Man20, Glu40Gal30Man30, Glu20Gal40Man40, Glu10Gal45Man45, Glu5Gal90Man5, Glu10Gal80Man10, Glu20Gal60Man20, Glu30Gal40Man30, 20Man40, Glu45Gal10Man45, Glu5Gal5Man90, Glu10Gal10Man80, Glu20Gal20Man60, Glu30Gal30Man40, Glu40Gal40Man20 and Glu45Gal45Man10.

In some embodiments, the glycan polymer preparation comprises a mixture of four glycan units A, B, C and D independently selected from i) to vii) above, where A, B, C and D may be selected from the same or from a different group i) to vii) and where A, B, C and D can be selected in any desired ratio (for example, anywhere between 1-99% of A, 1-99% of B, 1 -99% C, 1-99% D, not exceeding 100%).

In some embodiments, the glycan polymer preparation comprises a mixture of five glycan units A, B, C, D and E independently selected from i) to vii) above, where A, B, C, D and E may be selected from among the same or a different group i) to vii) and where A, B, C, D and E can be selected in any desired ratio (for example, anywhere between 1-99% of A, 1-99 % of B, 1-99% of C, 1-99% of D, 1-99% of E, without exceeding 100%).

In some embodiments, glycan polymer preparations are provided, wherein at least one glycan subunit is selected from the group consisting of a glucose, a galactose, an arabinose, a mannose, a fructose, a xylose, a fucose and a rhamnose. .

In some embodiments, the glycan polymer preparation comprises a desired mixture of two different monosaccharide glycan subunits, such as a mixture of, for example, glucose and galactose, glucose and arabinose, glucose and mannose, glucose and fructose, glucose and xylose, glucose and fucose, glucose and rhamnose, galactose and arabinose, galactose and mannose, galactose and fructose, galactose and xylose, galactose and fucose, and galactose and rhamnose, arabinose and mannose, arabinose and fructose, arabinose and xylose, arabinose and fucose , and arabinose and rhamnose, mannose and fructose, mannose and xylose, mannose and fucose, and mannose and rhamnose, fructose and xylose, fructose and fucose, and fructose and rhamnose, xylose and fucose, xylose and rhamnose, and fucose and rhamnose, for example in a ratio of 1:1, 1:2, 1:3, 1:4 or 1:5 or the inverse ratio thereof, or in a ratio of 1:1, 1:2, 1:3, 1 :4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:14, 1:16, 1:18, 1:20, 1:25 , 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1 :90, or 1:100 or the inverse ratio thereof.

In some embodiments, the glycan polymer preparation comprises a desired mixture of three different monosaccharide glycan subunits, such as a mixture of, for example, for glycan preparations containing glucose, glucose, galactose and arabinose; glucose, galactose and mannose; glucose, galactose and fructose; glucose, galactose and xylose; glucose, galactose and fucose, glucose, galactose and rhamnose; glucose, arabinose and mannose; glucose, arabinose and fructose; glucose, arabinose and xylose; glucose, arabinose and fucose; glucose, arabinose and rhamnose; glucose, mannose and fructose; glucose, mannose and xylose; glucose, mannose and fucose; glucose, mannose and rhamnose; glucose, fructose and xylose; glucose, fructose and fucose; glucose, fructose and rhamnose; glucose, fucose and rhamnose, for example in a ratio of 1:1:1, 1:2:1, 1:3:1, 1:4:1, 1:5:1, 1:1:2, 1: 2:2, 1:3:2, 1:4:2, 1:1:3, 1:2:3, 1:3:3, 1:1:4, 1:2:4, 1:1: 5, 1:2:5, etc., or in a ratio of 1:1:1, 1:2:1, 1:3:1, 1:4:1, 1:5:1, 1:6 :1, 1:7:1, 1:8:1, 1:9:1, 1:10:1, 1:12:1, 1:14:1, 1:16:1, 1:18:1 , 1:20:1, 1:1:2, 1:2:2, 1:3:2, 1:4:2, 1:5:2, 1:6:2, 1:7:2, 1 :8:2, 1:9:2, 1:10:2, 1:1:3, 1:2:3, 1:3:3, 1:4:3, 1:5:3, 1:6 :3, 1:7:3, 1:8:3, 1:9:3, 1:10:3, 1:1:4, 1:2:4, 1:3:4, 1:4:4 , 1:5:4, 1:6:4, 1:7:4, 1:8:4, 1:9:4, 1:10:4, 1:1:5, 1:2:5, 1 :3:5, 1:4:5, 1:5:5, 1:6:5, 1:7:5, 1:8:5, 1:9:5, 1:10:5, etc.

In some embodiments, the glycan polymer preparation does not comprise N-acetylgalactosamine or N-acetylglucosamine. In some embodiments, the glycan preparation does not comprise sialic acid. In some embodiments, the glycan polymer preparation does not comprise a lipid or fatty acid. In some embodiments, the glycan polymer preparation does not comprise an amino acid.

Furanosa.pyranose

In some embodiments, glycan polymer preparations are provided where at least one glycan subunit is a furanose sugar. In some embodiments, glycan preparations are provided where at least one glycan subunit is a pyranose sugar. In some embodiments, the glycan polymers comprise mixtures of furanose and pyranose sugars. In some embodiments, the furanose:pyranose sugar ratio in a preparation is about 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0. 6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.2:1, 1.5:1, 1.7:1, 2:1, 2, 2:1, 2.5:1, 2.7:1, 3:1, 4:1, 5:1 or about 6:1 or the sugar ratio of furanose:pyranose in a preparation is about 7:1 , 8:1, 9:1 or approximately 10:1.

In some embodiments, the glycan polymer preparation comprises substantially all furanose or pyranose sugar, optionally comprising 1%, 2%, 3%, 4% 5%, 6%, 7%, 8%, 9%, 10% , 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% of the respective other sugar.

In some embodiments, the glycan polymer preparation comprises substantially all pyranose sugar and no more than about 0.1%, 02%, 0.5%, 1%, 2%, 3%, 4% or no more than 5% glycan units in the preparation in the form of furanose. In some embodiments, no more than 3%, 2%, or no more than 1% of monomeric glycan units in the preparation are in the form of furanose.

You go out

In some embodiments, the glycan polymer preparation comprises a glycan subunit or plurality of glycan subunits present in a salt form (e.g., a pharmaceutically acceptable salt form), such as, for example, a hydrochloride salt, hydroiodide, hydrobromide, phosphate, sulfate, methanesulfate, acetate, formate, tartrate, malate, citrate, succinate, lactate, gluconate, pyruvate, fumarate, propionate, aspartate, glutamate, benzoate, ascorbate.

Derivatization

If desired, the monosaccharide or oligosaccharide glycan subunits of the glycans are further substituted or derivatized, for example, the hydroxyl groups may be etherified or esterified. For example, glycans (e.g. oligo- or polysaccharide) may contain modified saccharide units, such as 2'-deoxyribose where a hydroxyl group is removed, 2'-fluororribose where a hydroxyl group is replaced with a fluorine, or N-acetylglucosamine, a nitrogen-containing form of glucose (e.g., 2'-fluororibose, deoxyribose, and hexose). The degree of substitution (DS, average number of hydroxyl groups per glycosyl unit) can be 1, 2 or 3, or other suitable DS. In some embodiments, 1%, 2%, 3%, 4% 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16 %, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85% 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the glycan subunits are substituted or derivatized. In some embodiments, the degree of substitution varies between the subunits, for example, a certain percentage is not derivatized, has a DS of 1, has a DS of 2, or has a DS of 3. Any desired mixture can be generated, for example the 0-99% of subunits are not derivatized, 0-99% of subunits have a DS of 1, 0-99% of subunits have a DS of 2, and 0-99% of subunits have a DS of 3, the total constituting 100%. The degree of substitution can be controlled by adjusting the average number of moles of substituent added to a glycosyl moiety (molar substitution (MS)). The distribution of substituents along the length of the glycan oligo- or polysaccharide chain can be controlled by adjusting the reaction conditions, the type of reagent, and the magnitude of the substitution. In some embodiments, the monomeric subunits are substituted with one or more of an acetate ester, sulfate half-ester, phosphao ester, or a cyclic pyruvyl acetal group. Solubility

In some embodiments, the glycan polymers in a preparation are highly soluble. In some embodiments, the glycan polymer preparations can be concentrated to at least 55 Brix, 65 Brix, 60 Brix, 65 Brix, 70 Brix, 75 Brix, 80 Brix or at least 85 Brix without obvious solidification or crystallization at 23 ° C ( final solubility limit). In some embodiments, the glycan polymer preparations are concentrated to at least about 0.5 g/ml, 1 g/ml, 1.5 g/ml, 2 g/ml, 2.5 g/ml, 3 g/ml. ml, 3.5 g/ml or at least 4 g/ml without obvious solidification or crystallization at 23 °C (final solubility limit).

In some embodiments, the glycan polymer preparations (e.g., oligosaccharides) are branched, e.g., have an average DB of at least 0.01, 0.05, or 0.1, and have a final water solubility limit of at least 0.01, 0.05, or 0.1. less about 70 Brix, 75 Brix, 80 Brix or at least about 85 Brix at 23°C or is at least about 1 g/ml, 2 g/ml or at least about 3 g/ml.

In some embodiments, the glycan polymer preparation has a final solubility limit of at least 0.001 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.2 g/l, 0.3 g/l, 0.4 g/l, 0.5 g/l, 0.6 g/l, 0.7 g/l, 0.8 g/l, 0 .9 g/l, 1 g/l, 5 g/l, 10 g/l, 20 g/l, 30 g/l, 40 g/l, 50 g/l, 100 g/l, 200 g/l , 300 g/l, 400 g/l, 500 g/l, 600 g/l, 700 g/l, 800 g/l, 900 g/l, 1000 g/l in deionized water, or in a suitable buffer such such as phosphate-buffered saline, pH 7.4 or similar physiological pH) and at 20 °C. In some embodiments, the preparation of glycan polymers is greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 96%, greater than 97%. , more than 98%, more than 99% or more than 99.5% soluble without precipitation observed at a concentration of more than 0.001 g/l, 0.005 g/l, 0.01 g/l, 0.05 g/l , 0.1 g/l, 0.2 g/l, 0.3 g/l, 0.4 g/l, 0.5 g/l, 0.6 g/l, 0.7 g/l, 0.8 g/l, 0.9 g/l, 1 g/l, 5 g/l, 10 g/l, 20 g/l, 30 g/l, 40 g/l, 50 g/l, 100 g/l, 200 g/l, 300 g/l, 400 g/l, 500 g/l, 600 g/l, 700 g/l, 800 g/l, 900 g/l, 1000 g/l in water deionized, or in a suitable buffer such as, for example, phosphate-buffered saline, pH 7.4 or similar physiological pH) and at 20 ° C.

Sweetness

In some embodiments, the glycan polymer preparation has a desired degree of sweetness. For example, sucrose (table sugar) is the prototype of a sweet substance. Sucrose in solution has a sweetness perception rating of 1, and other substances are rated relative to this (for example, fructose is rated at 1.7 times the sweetness of sucrose). In some embodiments, the sweetness of the glycan polymer preparation ranges from 0.1 to 500,000 relative to sucrose. In some embodiments, the relative sweetness is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60 , 65, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 00 , 4000, 5000, 6000, 7000, 8000, 9000, 10000, 25000, 50000, 75000, 100000, 150000, 200000, 250000, 300000, 350000, 40000, 50,000, 500,000 or more than 500,000 in relation to sucrose (scored by sucrose as one). In some embodiments, the glycan polymer preparation is slightly sweet, or both sweet and bitter.

In some embodiments, the preparation of glycan polymers, for example a preparation that is substantially DP2+ or DP3+ (e.g., at least 80%, 90% or at least 95%, or a fractionated preparation of DP2+ or DP3+) , is substantially undetectable as sweet and the relative sweetness is about 0, 0.0001, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 , 0.6, 0.7, or approximately 0.8 relative to sucrose (sucrose scored as one).

Fermentability

In some embodiments, the glycan polymer preparations disclosed herein are examined to evaluate their fermentability. The fermentability of a glycan polymer is a function of the number or representation of hydrolyzable glycosidic bonds in the glycan species of the preparation. In some embodiments, fermentability is tested using a glycosidase enzyme or a glycosidase enzyme molecule described herein. A glycan polymer produced by the methods described herein, for example, using a glycosidase enzyme molecule, is believed to be a substrate for a glycosidase enzyme (e.g., that of a human intestinal microbe) that is closely related to the glycosidase enzyme molecule (e.g., they share a high degree of relevant sequence homology, the glycosidase enzyme molecule is a derivative of the glycosidase enzyme, they share the same origin (e.g., microbial origin, they share the same glycosidic functionality, are members of the CAZy family of glycoside hydrolase or glycoside transferase, etc.).

In some embodiments, the degree of fermentability of the glycan polymer preparation is 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, 4 minutes or less, 3 minutes or less. less, 2 minutes or less or 1 minute or less. In some embodiments, the digestibility of the glycan polymer preparation is 30 minutes or more, 45 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, or 10 hours or more, in which 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80 %, 90%, 95%, 97%, 99% of the glycans in the preparation have been fermented, e.g., decomposed, so that the glycan polymers in the preparation exhibit a reduction in average DP (e.g., DP = 5 at DP = 4) and/or a gain (or loss) of small molecular weight fractions (e.g. monomers, dimers, trimers) by conventional methodology (e.g. size exclusion chromatography). In some embodiments, the glycan polymers of the glycan polymer preparation comprise less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12% , 14%, 16%, 18%, 20%, 30%, 40%, or less than 50% of bonds that are hydrolyzable by a mammalian enzyme (e.g., amylase).

Suitable assays can be used to evaluate comparative fermentability (e.g., against a reference glycan) or to evaluate absolute digestibility.

Identification and analysis of glycan polymers

The following aspects and embodiments for identifying and analyzing glycan polymers are for illustrative purposes.

If desired, glycan polymer preparations can be characterized. For example, monomeric building blocks (e.g., composition of monosaccharide or glycan subunits), anomeric configuration of side chains, presence and location of substituent groups, degree of polymerization/molecular weight, and binding pattern can be identified by conventional methods known in the art, such as, for example, methylation analysis, reductive cleavage, hydrolysis, GC-MS (gas chromatography-mass spectrometry), MALDI-MS (matrix-assisted laser desorption/ionization -mass spectrometry), ESI-MS (electrospray ionization-mass spectrometry), HPLC (high-performance liquid chromatography with ultraviolet or refractive index detection), HPAEC-PAD (high-performance anion exchange chromatography with detection pulsed amperometric), CE (capillary electrophoresis), IR (infrared)/Raman spectroscopy and NMR (nuclear magnetic resonance) spectroscopy techniques. For polymers of crystalline consistency, the crystal structure can be resolved using, for example, solid-state NMR, FT-IR (Fourier transform infrared spectroscopy) and WAXS (high-angle X-ray scattering). DP, DP distribution and polydispersity can be determined, for example, by viscosimetry and SEC (SEC-HPLC, high performance size exclusion chromatography). Foreign groups, end groups and substituents can be identified, for example, using SEC with labeling, aqueous analysis, MALDI-MS, FT-IR and NMR. To identify the monomeric components of glycans, methods such as, for example, acid-catalyzed hydrolysis, HPLC (high-performance liquid chromatography) or GLC (gas-liquid chromatography) (after conversion to alditol acetates) can be used. ). To determine the linkages present in glycans, in one example, the polysaccharide is methylated with methyl iodide and a strong base in DMSO, hydrolysis is performed, reduction is achieved to partially methylated alditols, acetylation is performed to methylated alditol acetates and the analysis is carried out by GLC/MS (gas-liquid chromatography coupled to mass spectrometry). In some embodiments, partial depolymerization is carried out to determine the polysaccharide sequence using an acid or enzymes to determine the structures. The possible structures of the polysaccharide are compared with those of the hydrolytic oligomers and it is determined which of the possible structures could produce the oligomers. To identify the anomeric configuration, in one example, the intact polysaccharide or an oligosaccharide preparation is subjected to enzymatic analysis, for example, contacted with an enzyme that is specific for a particular type of attachment, for example, p-galactosidase. or a-glucosidase, etc., and NMR can be used to analyze the products.

For example, the degree of polymerization (DP) distribution (or average) of a glycan polymer preparation can be measured by injecting a sample with a concentration of, for example, 10-100 mg/ml into an Agilent 1260 BioPure HPLC instrument ( or similar) equipped with a BioRad Aminex HPX-42A 7.8x300 mm column (or similar) and an RI detector as described, for example, in Gómezet al. (Purification, Characterization, and Prebiotic Properties of Pectic Oligosaccharides from Orange Peel Wastes, J Agric Food Chem, 2014, 62:9769). Alternatively, a sample concentration can be injected into a Dionex ICS5000 (or similar) HPLC instrument equipped with a Dionex CarboPac PA1 4x250 mm column (or similar) and a PAD detector as described, for example, in Holcket al. ,(Feruloylated and nonferuloylated arabino-oligosaccharides from sugar beet pectin selectively stimulate the growth of bifidobacterium spp. in human fecal in vitro fermentations, Journal of Agricultural and Food Chemistry, 2011, 59(12), 6511-6519). Integration of the resulting spectrum in comparison to a standstill solution of oligomers allows determination of the average DP.

The molecular weight distribution can be measured, for example, by MALDI mass spectrometry. Oligosaccharide concentration can be measured with a Mettler-Toledo (or similar) sugar refractometer with the final value fitted against a standardized curve to account for refraction differences between monomers and oligomers.

The distribution of glycoside regiochemistry can be characterized, for example, by a variety of 2D NMR techniques including COSY, HMBC, HSQC, DEPT and TOCsY analysis using conventional pulse sequences and a 500 MHz Bruker spectrometer. Peaks can be assigned by correlation with the spectra of naturally occurring polysaccharides with known regiochemistry.

Monomeric compositions of oligomers can be measured, for example, by the complete hydrolysis method in which a known amount of oligomer is dissolved in a strong acid at elevated temperature and sufficient time is allowed for complete hydrolysis to occur. The concentration of individual monomers can then be measured by the HPLC or GC methods described herein and known in the art to achieve relative abundance measurements as in Holcket al. Absolute quantities can be measured by adding to the HPLC sample a known amount active pattern of the selected detector to avoid overlap with any of the critical signals.

The degree of branching in any given population can be measured by the method of methylation analysis established, for example, by Hakomori (J. Biochem. (Tokyo), 1964, 55, 205). With these data, identification of possible repeat units can be established by combining data from total hydrolysis, average DP, and methylation analysis and comparing them with the DEPT NMR spectrum. Correlating the number of anomeric carbon signals with this data indicates whether a regular repeat unit is required to satisfy the collected data as demonstrated, for example, in Harding, et al. (Carbohydr. Res. 2005, 340, 1107 ).

The molar percentage of species with a degree of polymerization (DP) of n (denoted here as DP(n)) in a population is determined by high-performance liquid chromatography (HPLC), for example, on an instrument of the Agilent 1260 BioInert series equipped with a refractive index (RI) detector and a variety of columns familiar to those skilled in the art using water as the mobile phase. Columns are selected from chemistries including, but not limited to, HILIC, metal coordination, and aqueous size exclusion chromatography that best isolate species of interest. The molar % of DP(n) is determined by the formula:

% DP(n) = 100 * AUC [DP(n)] / AUC [DP(total)],

where AUC is defined as the area under the curve for the species of interest as determined by calibration with known standards. The molar percentage of glycosidic bond isomers (% alpha and % beta) are determined by nuclear magnetic resonance (NMR) spectroscopy using a variety of 2D techniques familiar to those skilled in the art. The alpha and beta isomers can be distinguished, for example, by their different shift in the NMR spectrum and the molar percentage is determined by the formula:

% (n-glycosidic isomer) glycosidic bonds = 100* AUC [shift (n-isomer)] / AUC [shift (alpha isomer beta isomer)],

where AUC is defined as the area under the curve at a specific displacement value known to represent the desired isomer n. The molar percentage of regiochemical isomers is determined analogously using the formula:

% (n-regioisomer) of regioisomers = 100* AUC [shift (n-regioisomer)] / AUC [shift (all regioisomers)].

The relative percentage of monomeric sugars constituting the oligomeric population is determined, for example, by total acid digestion of the oligomeric sample followed by conversion to alditol acetate followed by gas chromatography (GC) analysis of the resulting monomeric solutions in comparison to GC of known patterns. The molar percentage of monomer (n), where n can be any sugar, is determined by the formula:

% (n sugar) = 100* AUC [n sugar] / AUC [total of all monomeric sugars].

In some embodiments, the solubility of the glycan polymer preparation can be controlled, for example, by selecting the charge, structure (e.g., DP, degree of branching) and/or derivatization of the glycan units.

Glycan polymer preparations consisting of one type of sugar unit uniformly linked in linear chains are usually insoluble in water at 23 °C even when the glycans have a low molecular weight with degrees of polymerization (DP) between 20 and 30 The degree of solubility of glycan polymers can be adjusted, for example, by introducing (1->6) linkages and alternating glycosidic linkages into the glycans. The additional degrees of freedom provided by the rotation around the C-5 to C-6 bonds provide higher solution entropy values. Homoglycans with two types of sugar linkages or heteroglycans composed of two types of sugars are generally more soluble than homogeneous polymers. Ionization of linear homoglycans can add solubility, for example, to that of gels. The viscosity of solutions often depends on the tertiary structures of the glycans.

Formulations and dosages of glycan polymers

The following aspects and embodiments for preparing glycan polymers with specific formulations are for illustrative purposes.

Also provided herein are methods for producing compositions (e.g., pharmaceutical compositions) comprising a glycan polymer preparation that meets one or more, two or more, three or more, or four or more of the characteristics of the preparations. described in this document (including criteria (i)-(v) above). In particular, the methods include providing a glycan polymer preparation and acquiring the value(s) for one or more, two or more, or three or more characteristics of the preparation, including, for example, i) the degree of polymerization (DP), ii) the average degree of branching (DB, branch points per residue), iii) the ratio of alpha-glycosidic to beta-glycosidic bonds, iv) the identity of the glycan subunits and v) the ratio of glycan subunits, and producing a pharmaceutical composition comprising a glycan polymer preparation if the desired or predetermined criteria of the preparation are met within a desired deviation range.

Methods for formulating the glycan polymer preparation into a pharmaceutical composition, medical food or dietary supplement are known in the art and may include one or more, two or more, three or more, or four or more of the following steps: (i ) formulate the preparation to give a pharmaceutical product, (ii) package the preparation, (iii) label the packaged preparation and (iv) sell or offer for sale the packaged and labeled preparation. Formulation of the glycan polymer preparation to give a pharmaceutical product is known in the art and may include one or more, two or more, three or more, or four or more of the following steps: (i) removing unwanted constituents of the preparation, (ii) reduce the volume of the preparation, (iii) sterilize the preparation, (iv) mix the preparation with a pharmaceutically acceptable excipient or carrier, (v) mix the preparation with a second drug or pharmaceutical agent, ( vi) formulating the preparation in a suitable consistency, such as, for example, a dilute aqueous solution, a syrup or a solid, (vii) formulating the preparation in a suitable dosage form, for example, in a tablet, lozenge or capsule .

In some embodiments, the glycan polymer preparation is subjected to further processing to produce either glycan polymer syrup or powder. For example, in one variation, the glycan polymer preparation is concentrated to form a syrup. Any suitable method known in the art can be used to concentrate a solution, such as the use of a vacuum evaporator. In another variation, the glycan polymer preparation is spray dried to form a powder. Any suitable method known in the art can be used to spray dry a solution to form a powder. Provided herein are pharmaceutical compositions, medical foods and dietary supplements comprising glycan polymer preparations. Optionally, pharmaceutical compositions, medical foods and dietary supplements comprising glycan polymer preparations further comprise a second agent, for example, a prebiotic substance and/or a probiotic bacteria. In some embodiments, pharmaceutical compositions and medical foods and dietary supplements comprising glycan polymer preparations further comprise a micronutrient. In some embodiments, pharmaceutical compositions and medical foods and dietary supplements comprising glycan polymer preparations do not contain a prebiotic substance. In some embodiments, pharmaceutical compositions and medical foods and dietary supplements comprising glycan polymer preparations do not contain a probiotic bacteria. Furthermore, optionally, pharmaceutical compositions and medical foods and dietary supplements comprising glycan polymer preparations comprise one or more excipients or carriers, including diluents, binders, disintegrants, dispersants, lubricants, glidants, stabilizers, surfactants, flavoring agents and colorants.

In some embodiments, pharmaceutical compositions and medical foods and dietary supplements comprising glycan polymer preparations (and kits comprising the same) comprise one or more micronutrients. In some embodiments, the micronutrient is selected from the group consisting of a trace element, choline, a vitamin, and a polyphenol. In some embodiments, the micronutrient is a trace element. Trace elements suitable as micronutrients include, but are not limited to, boron, cobalt, chromium, calcium, copper, fluorine, iodine, iron, magnesium, manganese, molybdenum, selenium and zinc. In some embodiments, the micronutrient is a vitamin. In some embodiments, the micronutrient is a polyphenol. Furthermore, if desired, pharmaceutical compositions and medical foods and dietary supplements comprising glycan polymer preparations may comprise therapeutically active agents, prebiotic substances and/or probiotic bacteria. Alternatively or in addition, the therapeutically active agents, prebiotic substances and/or probiotic bacteria may be administered separately (e.g., before, simultaneously with or after the administration of the glycan polymers) and not as part of the pharmaceutical composition or medical food. or dietary supplement (e.g., as a coformulation) of glycan polymers. In some embodiments, the pharmaceutical compositions or medical foods or dietary supplements comprising glycan polymer preparations are administered in combination with a recommended or prescribed diet, for example, a diet rich in foods containing probiotics and/or prebiotics, such as determined by a doctor or other health professional. Therapeutically active agents, prebiotic substances and/or probiotic bacteria may be administered to modulate the subject's gut microbiome. In some embodiments, the combined effect (e.g., on the number or intensity of microbial, genomic or functional changes) is additive. In other embodiments, the combined effect (for example, on the number or intensity of microbial, genomic or functional changes) is synergistic.

In some embodiments, the pharmaceutical compositions and medical foods and dietary supplements comprising glycan polymer preparations described herein further comprise a prebiotic substance or a preparation thereof.

In some embodiments, prebiotics may be administered to a subject receiving the pharmaceutical compositions or medical foods or dietary supplements comprising glycan polymer preparations described herein. Prebiotics are non-digestible substances that, when consumed, can provide a beneficial physiological effect on the host by selectively stimulating the growth or favorable activity of a limited number of indigenous bacteria in the intestine (Gibson G R, Roberfroid M B. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr. June 1995; A prebiotic such as a dietary fiber or a prebiotic oligosaccharide (e.g., crystalline cellulose, wheat bran, oat bran, corn fiber, soybean fiber, beet fiber and the like) may further stimulate the growth of probiotic bacteria and/or or commensals in the intestine by providing a fermentable dose of carbohydrates to bacteria and increasing the levels of those microbial populations (e.g., lactobacilli and bifidobacteria) in the gastrointestinal tract. Prebiotics include, but are not limited to, various galactans and carbohydrate-based gums, such as psyllium, guar, carrageenan, gellan, lactulose and konjac. In some embodiments, the prebiotic is one or more of galactooligosaccharides (GOS), lactulose, raffinose, stachyose, lactosucrose, fructooligosaccharides (FOS, for example, oligofructose or oligofructan), inulin, isomaltooligosaccharide, xylooligosaccharides (XOS), paratinose oligosaccharide, oligosaccharides isomaltose (IMOS), transgalactosylated oligosaccharides (e.g., transgalactooligosaccharides), transgalactosylate disaccharides, soybean oligosaccharides (e.g., soybean oligosaccharides), chitosan oligosaccharides (chiose), gentiooligosaccharides, soybean and pectin oligosaccharides, glucooligosaccharides, pecticooligosaccharides, polycondensates of palatinose, fructose anhydride III, sorbitol, maltitol, lactitol, polyols, polydextrose, linear and branched dextrans, pullulan, hemicelluloses, reduced paratinose, cellulose, betaglucose, betagalactose, betafructose, verbascose, galactinol, xylan, inulin, chitosan, betaglucan, gum guar, gum arabic, pectin, high sodium alginate and lambda carrageenan, or mixtures thereof. Examples of suitable probiotics include, but are not limited to, organisms classified as genera Bacteroides, Blautia, Clostridium, Fusobacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, Akkermansia, Faecalibacterium, Roseburia, Prevotella, Bifidobacterium, Lactobacillus, Bacillus, Enterococcus, Escherichia, Streptococcus, Saccharomyces, Streptomyces, and Christensenellaceae family. Non-exclusive examples of probiotic bacteria that can be used in the methods and compositions described herein include L. acidophilus, Lactobacillus species, such as L. crispatus, L. casei, L. rhamnosus, L. reuteri, L. fermentum, L. plantarum, L. sporogenesyL. bulgaricus, as well as Bifidobacterum species, such as B. lactis, B. animalis, B. bifidum, B. longum, B. adolescentis, and B. infantis. As probiotics for administration to the intestine, yeasts such as Saccharomyces boulardii are also suitable, for example, by means of oral dosage forms or foods. In some embodiments, the taxon of probiotic bacteria is not Bifidobacterium. In some embodiments, the taxon of probiotic bacteria is not Lactobacillus. Bacteria beneficial for modulation of the gastrointestinal microbiota may include bacteria that produce organic acids (lactic and acetic acids) or that produce cytotoxic or cytostatic agents (to inhibit pathogen growth), such as, for example, hydrogen peroxide (H2O2) and bacteriocins. Bacteriocins are small antimicrobial peptides that can kill closely related bacteria or exhibit a broader spectrum of activity (e.g., nisin).

Beneficial bacteria may include one or more of the genus Akkermansia, Anaerophilum, Bacteroides, Blautia, Bifidobacterium, Butyrivibrio, Clostridium, Coprococcus, Dialister, Dorea, Fusobacterium, Eubacterium, Lachnospira, Lactobacillus, Phascolarctobacterium, Peptococcus, Peptostreptococcus, Prevotella, Roseburia, Ruminococcus, and Streptococcus In some embodiments, the probiotic or commensal bacteria include a or more of the bacteria listed in Table 2.

Prebiotic substances and probiotic strains that can be combined with the glycan polymers described herein to produce a composition or kit can be isolated at any level of purity by conventional methods and purification can be achieved by conventional means known to those skilled in the art. technique, such as distillation, recrystallization and chromatography. The cultured bacteria to be used in the composition are separated from the culture broth by any method including, without limitation, centrifugation, filtration or decantation. The cells separated from the fermentation broth are optionally washed with water, saline (0.9% NaCl) or with any suitable buffer. The wet cell mass obtained can be dried by any suitable method, for example, by freeze-drying. In some embodiments, the probiotic bacteria are freeze-dried vegetative cells. In some embodiments, spore preparations of sporulating probiotic bacteria are used.

In one embodiment, a glycan polymer preparation further comprises a prebiotic and a probiotic. In one embodiment, the pharmaceutical composition comprises probiotics whose viability has been partially attenuated (for example, a mixture comprising 10%, 20%, 30%, 40%, 50% or more non-viable bacteria), or probiotics consisting only in non-viable microbes. The compositions may further comprise microbial membranes and/or cell walls that have been isolated and purified from dead microbes. If desired, the probiotic organism can be incorporated into the glycan polymer preparation as a culture in water or other liquid or semi-solid medium in which the probiotic remains viable. In another technique, a lyophilized powder containing the probiotic organism can be incorporated into a particulate material or liquid or semi-solid material by mixing or combining it.

In some embodiments, pharmaceutical compositions and medical foods and dietary supplements comprising glycan polymer preparations further comprise a second therapeutic agent or a preparation thereof. In some embodiments, the therapeutic agent is an antibiotic, an antifungal agent, an antiviral agent, or an anti-inflammatory agent (e.g., a cytokine, a hormone, etc.).

The glycan polymer preparations described herein, other therapeutically active agents, prebiotic substances, micronutrients and probiotics can be combined or mixed in a single pharmaceutical composition or medical food or dietary supplement. In other embodiments, they may be contained in separate containers (and/or in various suitable unit dosage forms) but packaged together in one or more kits. In some embodiments, the preparations or compositions are not packaged or placed together. A physician may then administer the preparations or compositions together, for example before, concomitantly, or one after the other. In some embodiments, the preparations or compositions act synergistically in modulating the microbiota in a subject, for example, in the GI tract.

In some embodiments, a glycan polymer composition comprises between 0.1% and 100% glycan polymer preparation in w/w, w/v, v/v, or mol%. In another embodiment, a glycan polymer composition comprises about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13% %, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46% , 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63 %, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, 99.5% or 100% of the glycan polymer preparation in w/w, w/v, v/v or mol %. In one embodiment, a glycan polymer composition comprises about 1-90%, about 10-90%, about 20-90%, about 30-90%, about 40-90%, about 40-90%, about 40-90%, about 40-90%, about 40-90%. 80%, approximately 40-70%, approximately 40-60%, approximately 40-50%, approximately 50-90%, approximately 50-80%, approximately 50-70%, approximately 50-60 %, approximately 60-90%, approximately 60-80%, approximately 60-70%, approximately 70-90%, approximately 70-80%, approximately 70-90%, approximately 70-80% , approximately 80-90%, approximately 90-96%, approximately 93-96%, approximately 93-95%, approximately 94-98%, approximately 93-99% or approximately 90-100% of the preparation of glycan polymer in w/w, w/v, v/v or mol %. A composition comprising a glycan polymer preparation may optionally comprise one or more excipients or carriers. The glycan polymer composition may comprise from about 1% to about 90% of one or more excipients or carriers in w/w%, w/v, v/v or mol%. For example, the glycan polymer composition may comprise about 1-90%, 1-75%, 1-60%, 1-55%, 1-50%, 1-45%, 1-40%, 1-25 %, 1-15%, 1-10%, 10-90%, 10-75%, 10-60%, 10-55%, 10-50%, 10-45%, 10-40%, 10-25% , 10-15%, 15-90%, 15-75%, 15-60%, 15-55%, 15-50%, 15-45%, 15-40%, 15-25%, 25-90% , 25-75%, 25-60%, 25-55%, 25-50%, 25-45%, 25-40%, 40-90%, 40-75%, 40-60%, 40-55% , 40-50%, 40-45%, 45-90%, 45-75%, 45-60%, 45-55%, 45-50%, 50-90%, 50-75%, 50-60% , 50 55%, 55-90%, 55-75%, 55-60%, 60-90%, 60-75%, 75-90% of one or more excipients or carriers in w/w, w/v, v/v or molar %.

Medical foods

Also provided herein are preparations of glycan polymers formulated as medical food. Any glycan polymer preparation described herein can be formulated as medical food, as well as pharmaceutical compositions comprising glycan polymer preparations.

A medical food is defined in section 5(b)(3) of the Orphan Drug Act (21 U.S.C.

360ee(b)(3)). Medical foods are formulated to be consumed (oral intake) or administered enterally (e.g., nasogastric feeding/tube) under medical supervision, such as by a physician. It is intended for the specific dietary treatment of a disease or condition, such as dysbiosis or a GI disease. Medical foods as used herein do not include foods that are simply recommended by a doctor as part of an overall diet to manage symptoms or reduce the risk of a disease or condition. Medical foods comprising a preparation of glycan polymers are foods that are synthetic food products (for example, formulated and/or processed products, such as formulated for partial or exclusive feeding of a patient by oral intake or enteral tube feeding). and that are not naturally produced used in a natural state.

In some embodiments, the subject has a limited or impaired ability to ingest, digest, absorb or metabolize common food products or certain nutrients. In other embodiments, the subject has other medically determined special nutritional requirements, the dietary management of which cannot be achieved by modification of the normal diet alone. Medical foods comprising a preparation of glycan polymers are administered to a subject in need thereof under medical supervision (which may be active and continuous) and the subject usually receives instructions on the use of the medical food. Medical foods may comprise one or more food additives, color additives, GRAS excipients and other agents or substances suitable for medical foods. Medical food preparations can be nutritionally complete or incomplete formulas.

Dietary supplements

Any glycan polymer preparation described herein may be formulated as a dietary supplement, for example, for use in a method described herein. Dietary supplements are regulated by the Dietary Supplement Health and Education Act (DSHEA) of 1994. A dietary supplement is a product taken by mouth that contains a “dietary ingredient” intended to supplement the diet. "Dietary ingredients" in these products may include, in addition to a glycan polymer preparation described herein, one or more of: vitamins, minerals, herbs or other botanicals, amino acids, and substances such as enzymes, organ tissues, glandular and metabolites. Dietary supplements can also be extracts or concentrates and can be found in many forms, such as tablets, capsules, softgels, gel caps, liquids or powders. They may also come in other forms, such as a bar, but if they are, the information on their label should not represent the product as a conventional food or a single element of a meal or diet. DSHEA requires that each supplement be labeled as a dietary supplement and not a general food.

food ingredient

Any glycan polymer preparation described herein may be formulated as a food ingredient or food additive, for example, for use in a method described herein. Food ingredients may be generally recognized as safe (GRAS) or may require FDA clearance. Glycan polymer preparations can be added to any desired food, for example, beverages (including, for example, fruit juices), dairy products (for example, milk, yogurt, cheese), cereals (any cereal products), bread, spreads, etc.

The glycan polymer preparations described herein may be formulated in any suitable dosage form, for example, for nasal, oral, rectal or gastric administration. In some embodiments, the glycan polymer preparations described herein may be formulated for enteral administration. In some embodiments, the glycan polymer preparations described herein may be formulated for tube feeding (e.g., nasogastric, oral-gastric, or gastric feeding). The dosage forms described herein can be manufactured using processes known to those skilled in the art.

The dosage form may be a packet, such as any individual container containing a glycan polymer preparation in the form of, for example, a liquid (wash/rinse), a gel, a cream, an ointment, a powder, a tablet, a pill, a capsule, a reservoir, a single-use applicator, or a medical device (for example, a syringe). For example, also provided is an article of manufacture, such as a container comprising a unit dosage form of the glycan polymer preparation, and a label containing instructions for the use of such glycan polymer.

Forms of the compositions that can be used orally include tablets, snap-fit capsules made of gelatin, as well as sealed soft capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Tablets may be prepared by compression or molding, optionally with one or more accessory ingredients. Compression tablets can be prepared by compressing in a suitable machine the active ingredient in a free-flowing form, such as powder or granules, optionally mixed with binders (for example, povidone, gelatin, hydroxypropylmethylcellulose), inert diluents, preservatives, antioxidants, disintegrants (for example, sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethylcellulose) or lubricating, surfactant or dispersing agents. Molded tablets can be prepared by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. Optionally, the tablets may be coated or scored and may be formulated to provide a slow or controlled release of the active ingredient they contain. Optionally, the tablets may be provided with an enteric coating, to provide release in parts of the intestine (eg, colon, lower intestine) other than the stomach. All formulations for oral administration may be in dosages suitable for such administration. The snap-fit capsules may contain the active ingredients in admixture with fillers such as lactose, binders such as starches and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds and/or other agents (for example, prebiotics or probiotics) can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin or liquid polyethylene glycols. Additionally, stabilizers can be added. Sugar-coated tablet cores with suitable coatings are provided. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol or titanium dioxide, shellac solutions and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to tablets or sugar-coated tablets for identification or to characterize different dose combinations of active compounds. Formulations for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with a water-soluble carrier, such as polyethylene glycol or an oily medium, for example peanut oil, liquid paraffin or olive oil.

In one embodiment, a provided glycan polymer preparation includes a soft capsule formulation. A soft capsule may contain a gelatin-based shell surrounding a liquid filling. The shell may be made of gelatin, plasticizer (e.g., glycerin and/or sorbitol), modifier, water, color, antioxidant or flavoring. The covering can be made of starch or carrageenan. The outer layer may be enteric coated. In one embodiment, a soft capsule formulation may include a water- or oil-soluble filling solution, or a suspension of a composition coated by a gelatin layer.

Solid formulations for oral use may comprise an enteric coating, which may control the location at which a glycan polymer preparation is absorbed in the digestive system. For example, an enteric coating can be designed so that a glycan polymer preparation does not dissolve in the stomach but travels to the small intestine, where it dissolves. An enteric coating may be stable at a low pH (such as in the stomach) and may dissolve at a higher pH (for example, in the small intestine). Material that can be used in enteric coatings includes, for example, alginic acid, cellulose acetate phthalate, plastics, waxes, shellac and fatty acids (for example, stearic acid, palmitic acid).

Formulations for oral use may also be presented in liquid dosage form. Liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives, such as suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate , gum arabic; non-aqueous vehicles (which may include edible oils), for example almond oil, oily esters such as glycerin, propylene glycol or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid and, if desired, conventional flavoring or coloring agents. In some embodiments, the liquid formulations may comprise, for example, an agent in the form of water in solution and/or suspension; and a carrier comprising polyethoxylated castor oil, alcohol and/or a polyoxyethylated sorbitan monooleate with or without flavoring. Each dosage form may comprise an effective amount of a glycan polymer and may optionally comprise pharmaceutically inert agents, such as excipients, vehicles, fillers, binders, disintegrants, pH adjusting substances, buffers, solvents, solubilizing agents, sweeteners, agents. conventional dyes and any other inactive agents that may be included in pharmaceutical dosage forms for administration. Examples of such carriers and additives can be found in Remington's Pharmaceutical Sciences, 17th edition (1985).

The pharmaceutical compositions provided herein may be in unit dosage forms or multiple dosage forms. A unit dosage form, as used herein, refers to a physically distinct unit suitable for administration to a human being in need. In one embodiment, the unit dosage form is provided in a package. Each unit dose may contain a predetermined amount of active ingredient(s) sufficient to produce the desired therapeutic effect, in association with other pharmaceutical carriers or excipients. Examples of unit dosage forms include, but are not limited to, ampoules, syringes, and individually packaged tablets and capsules. Unit dosage forms may be administered in fractions or multiples thereof. A multiple dosage form is a plurality of identical unit dosage forms packaged in a single container, which may be administered in segregated unit dosage form. Examples of multiple dosage forms include, but are not limited to, vials, tablet or capsule bottles, or pint or gallon bottles. In another embodiment, multiple dosage forms comprise different pharmaceutically active agents. For example, a multiple dosage form may be provided comprising a first dosage element comprising a composition comprising a glycan polymer and a second dosage element comprising a prebiotic, a therapeutic agent and/or a probiotic, which may be in a modified release form. In this example, a pair of dosing elements can form a single unit dosage. In one embodiment, a kit is provided comprising multiple unit dosages, wherein each unit comprises a first dosage element comprising a composition comprising a glycan polymer preparation and a second dosage element comprising a probiotic, a pharmaceutical agent , a prebiotic or a combination thereof, which may be in a modified release form. In another embodiment, the kit further comprises a set of instructions.

In some embodiments, the unit dosage form comprises between about 1 mg and about 100 g of the glycan polymer preparation (e.g., a glycan polymer disclosed herein). For example, the unit dosage form may comprise from about 50 mg to about 50 g, from about 500 mg to about 50 g, from about 5 g to about 50 g, from about 100 mg to about 100 g, from about 1 g to about 100 g, about 10 g to about 100 g, about 1 g to about 10 g, about 1 g to about 20 g, about 1 g to about 30 g, about 1 g to about 40 g, about 1 g to about 50 g , from about 1 g to about 60 g, from about 1 g to about 70 g, from about 1 g to about 80 g, from about 1 g to about 90 g, from about 1 g to about 100 g, from about 1 g to about 150 g, about 1 g to about 200 g of the glycan polymer.

In other embodiments, the unit dosage form comprises between about 0.001 ml and about 1000 ml of the glycan polymer (e.g., a glycan polymer disclosed herein). For example, the unit dosage form may comprise from about 0.001 ml to about 950 ml, from about 0.005 ml to about 900 ml, from about 0.01 ml to about 850 ml, from about 0.05 ml to about 800 ml, from about 0.075 ml to about 750 ml, from about 0.1 ml to about 700 ml, from about 0.25 ml to about 650 ml, from about 0.5 ml to about 600 ml, from about 0.75 ml to about 550 ml, from about 1 ml to about 500 ml, from about 2.5 ml to about 450 ml, from about 5 ml to about 400 ml, from about 7.5 ml to about 350 ml, from about 10 ml to about 300 ml, from about 12.5 ml to about 250 ml, from about 15 ml to about 200 ml, from about 17.5 ml to about 150 ml, from about 20 ml to about 100 ml or from about 25 ml to about 75 ml of the glycan polymer.

In certain embodiments, the unit dosage form comprises from about 0.001 ml to about 10 ml, from about 0.005 ml to about 7.5 ml, from about 0.01 ml to about 5 ml, from about 0.05 ml to about 2 .5 ml, from about 0.1 ml to about 1 ml, from about 0.25 ml to about 1 ml or from about 0.5 ml to about 1 ml of the glycan polymer. In other embodiments, the unit dosage form comprises from about 0.01 ml to about 10 ml, from about 0.025 ml to about 7.5 ml, from about 0.05 ml to about 5 ml, or from about 0.1 ml to approximately 2.5 ml of the glycan polymer. In other embodiments, the unit dosage form comprises from about 0.1 ml to about 10 ml, from about 0.25 ml to about 7.5 ml, from about 0.5 ml to about 5 ml, from about 0.5 ml. ml to about 2.5 ml or from about 0.5 ml to about 1 ml of the glycan polymer.

In some embodiments, the unit dosage form, e.g., a tablet, capsule (e.g., a hard capsule, a snap-fit capsule, or a soft capsule), or softgel, has a body length of between about 0 .1 inch and about 1.5 inches (e.g., between about 0.5 inches and about 1 inch) or between about 5 mm and about 50 mm (e.g., between about 10 mm and about 25 mm). In some embodiments, the unit dosage form, e.g., a tablet, capsule (e.g., a hard capsule, snap-fit capsule, or soft capsule), or soft gel capsule, has an outer diameter of about 0.05 inches to about 1 inch (e.g., about 0.1 inch to about 0.5 inch) or about 1 mm to about 25 mm (e.g., about 5 mm to about 10 mm).

Each unit dosage form of the glycan polymer may have a caloric value of between about 0.01 kcal and about 1000 kcal. For example, the unit dosage form may have a caloric value of about 0.01 kcal to about 100 kcal, about 0.05 kcal to about 50 kcal, about 0.1 kcal to about 10 kcal, about 0. 25 kcal to approximately 2.5 kcal, approximately 0.5 kcal to approximately 5 kcal, approximately 0.75 kcal to approximately 7.5 kcal, approximately 1 kcal to approximately 10 kcal, approximately 5 kcal to approximately 50 kcal or from about 10 kcal to about 100 kcal. In certain embodiments, the unit dosage form of the glycan polymer has a caloric value of between 10 kcal and about 500 kcal. In certain embodiments, the unit dosage form of the glycan polymer has a caloric value of between 1 kcal and about 100 kcal. In certain embodiments, the unit dosage form of the glycan polymer has a caloric value of between 0.1 kcal and about 10 kcal. In still other embodiments, the unit dosage form may have a caloric value of about 0.001 kcal to about 10 kcal, about 0.005 kcal to about 10 kcal, about 0.01 kcal to about 10 kcal, about 0.025 kcal to about 25 kcal, about 0.05 kcal to about 50 kcal, about 0.075 kcal to about 75 kcal, about 0.1 kcal to about 100 kcal, about 0.25 kcal to about 10 kcal, about 0.5 kcal to approximately 5 kcal, from approximately 0.25 kcal to approximately 25 kcal or from approximately 0.1 kcal to approximately 50 kcal approximately 1 kcal.

The unit dosage form of the glycan polymer may be formulated to dissolve in an aqueous solution (e.g., water, milk, juice and the like) and administered orally as a beverage, syrup, solution or suspension. For example, the unit dosage form of the glycan polymer may comprise a cube, packet, lozenge, pill, tablet, capsule, candy, powder, elixir or concentrated syrup formulated to dissolve in an aqueous solution prior to oral administration. In other embodiments, the unit dosage form of the glycan polymer may comprise a cube, packet, lozenge, pill, tablet, capsule, candy, powder, elixir or concentrated syrup formulated to dissolve in vivo, for example, in the mouth, the stomach, intestine or colon of the subject (for example, a human subject) after oral administration.

In some embodiments, the glycan polymer preparation is administered enterally. This preferably includes administration orally or via an oral or nasal tube (including nasogastric, nasojejunal, oral gastric or oral jejunal). In other embodiments, the administration includes rectal administration (including enema, suppository or colonoscopy).

The dosage forms described herein can be manufactured using processes known to those skilled in the art. For example, for the manufacture of tablets, an effective amount of a prebiotic can be uniformly dispersed in one or more excipients or additives, for example, using high shear granulation, low shear granulation, fluid bed granulation or mixing for direct compression. Excipients and additives include diluents, binders, disintegrants, dispersants, lubricants, glidants, stabilizers, surfactants, non-stick agents, sorbents, sweeteners and colorants, or a combination thereof. Diluents, also called fillers, can be used to increase the volume of a tablet so as to provide a practical size for compression. Non-limiting examples of diluents include lactose, cellulose, microcrystalline cellulose, mannitol, dry starch, hydrolyzed starches, powdered sugar, talc, sodium chloride, silicon dioxide, titanium oxide, dicalcium phosphate dihydrate, calcium sulfate, carbonate of calcium, alumina and kaolin. Binders can impart cohesive qualities to a tablet formulation and can be used to help a tablet remain intact after compression. Non-limiting examples of suitable binders include starch (including corn starch and pregelatinized starch), gelatin, sugars (for example, glucose, dextrose, sucrose, lactose and sorbitol), celluloses, polyethylene glycol, alginic acid, dextrin, casein, methylcellulose, waxes, natural and synthetic gums, for example, arabic, tragacanth, sodium alginate, gum arabic, xanthan gum and synthetic polymers such as polymethacrylates, poly(vinyl alcohols), hydroxypropylcellulose and polyvinylpyrrolidone. Lubricants can also facilitate tablet manufacturing; non-limiting examples thereof include magnesium stearate, calcium stearate, stearic acid, glyceryl behenate and polyethylene glycol. Disintegrants may facilitate disintegration of the tablet after administration, and non-limiting examples thereof include starches, alginic acid, cross-linked polymers such as, for example, cross-linked polyvinylpyrrolidone, croscarmellose sodium, sodium or potassium starch glycolate, clays, celluloses (for example, carboxymethylcelluloses (for example, carboxymethylcellulose).

(CMC), CMC-Na, CMC-Ca)), starches, gums and the like. Non-limiting examples of suitable sliders include silicon dioxide, talc and the like. Stabilizers can inhibit or slow down drug breakdown reactions, including oxidative reactions. Surfactants may also include and may be anionic, cationic, amphoteric or nonionic. Exemplary sweeteners may include stevia extract, aspartame, sucrose, alitame, saccharin, and the like. If desired, the tablets may also comprise non-toxic auxiliary substances such as pH buffering agents, preservatives, for example, antioxidants, wetting or emulsifying agents, solubilizing agents, coating agents, flavoring agents (for example, mint, cherry, anise , peach, apricot, licorice, raspberry, vanilla) and the like. Additional excipients and additives may include aluminum acetate, benzyl alcohol, butylparaben, butylated hydroxytoluene, calcium disodium EDTA, calcium hydrogen phosphate dihydrate, dibasic calcium phosphate, tribasic calcium phosphate, candelilla wax, carnauba wax, hydrogenated castor oil , cetylpyridine chloride, citric acid, colloidal silicone dioxide, copolyvidone, corn starch, cysteine HCl, dimethicone, disodium hydrogen phosphate, erythrosine sodium, ethylcellulose, gelatin, glycerin, glyceryl monooleate, glyceryl monostearate, glycine, HPMC phthalate , hydroxypropylcellulose, hydroxypropylmethylcellulose, hypromellose, red iron oxide or ferric oxide, yellow iron oxide, iron oxide or ferric oxide, magnesium carbonate, magnesium oxide, magnesium stearate, methionine, methacrylic acid copolymer, methylparaben, microcrystalline cellulose silicified, mineral oil, phosphoric acid, simple calcium phosphate, anhydrous calcium phosphate, polaxamer 407, polaxamer 188, simple polaxamer, polyethylene oxide, polyoxy stearate 140, polysorbate 80, potassium bicarbonate, potassium sorbate, potato starch , povidone, propylene glycol, propyleneparaben, propylparaben, retinyl palmitate, saccharin sodium, selenium, silica, silica gel, fumed silica, sodium benzoate, sodium carbonate, sodium citrate dihydrate, crossmellose sodium, sodium lauryl sulfate, metabisulfite sodium, sodium propionate, sodium starch, sodium starch glycolate, sodium stearyl fumarate, sorbic acid, sorbitol, sorbitan monooleate, pregelatinized starch, succinic acid, triacetin, triethyl citrate, vegetable stearin, vitamin A, vitamin E, vitamin C or a combination thereof. The amounts of these excipients and additives can be appropriately selected based on their relationship to other components and properties of the preparation and production method.

Immediate release formulations of an effective amount of a glycan polymer preparation may comprise one or more combinations of excipients that allow rapid release of a pharmaceutically active agent (such as from 1 minute to 1 hour after administration). Controlled release formulations (also known as sustained release (SR), extended release (ER, XR or XL), timed release or programmed release, controlled release (CR) or continuous release) refer to the release of a preparation of glycan polymer from a dosage form at a particular desired time after the dosage form is administered to a subject (e.g., a human subject).

In one embodiment, a controlled release dosage form begins its release and continues that release over an extended period of time. Release may occur almost immediately or may be sustained. The release may be constant, may increase or decrease over time, may be pulsed, may be continuous or intermittent, and the like. In one embodiment, controlled release dosing refers to the release of an agent from a composition or dosage form in which the agent is released according to a desired profile over an extended period of time. In one aspect, controlled release refers to the delayed release of an agent from a composition or dosage form in which the agent is released according to a desired profile in which the release occurs after a period of time. Suitable pharmaceutical carriers or vehicles for administration of the compounds provided herein include all carriers known to those skilled in the art to be suitable for the particular mode of administration. Furthermore, the compositions may have one or more components that do not alter the desired action, or with components that complement the desired action, or that have another action.

In a further aspect, the dosage form may be an effervescent dosage form. Effervescent means that the dosage form, when mixed with liquid, including water and saliva, gives off a gas. Some effervescent agents (or effervescent partners) release gas through a chemical reaction that occurs after exposure of the effervescent disintegrating agent to water or saliva in the mouth. This reaction may be the result of the reaction of a soluble acid source and a monocarbonate or alkali carbonate source. The reaction of these two general compounds produces carbon dioxide gas upon contact with water or saliva. An effervescent couple (or the individual acid and base separately) can be coated with an enteric coating or solvent protector to prevent premature reaction. Such a couple can also be mixed with previously lyophilized particles (such as a glycan polymer). Acid sources may be any that are safe for human consumption and may generally include dietary acids, acid antacids and hydrites such as, for example: citric, tartaric, amalic, fumeric, adipic and succinic. Carbonate sources include dry solid carbonate and bicarbonate salts such as sodium bicarbonate, sodium carbonate, potassium bicarbonate and potassium carbonate, magnesium carbonate and the like. Also included are reagents that release oxygen or other gases and are safe for human consumption. In one embodiment, citric acid and sodium bicarbonate are used.

In another aspect, the dosage form may be in candy (e.g., matrix) form, such as a lollipop or lozenge. In one embodiment, an effective amount of a glycan polymer is dispersed within a candy matrix. In one embodiment, the candy matrix comprises one or more sugars (such as dextrose or sucrose). In another embodiment, the candy matrix is a sugar-free matrix. The choice of a particular candy matrix is subject to wide variation. Conventional sweeteners (e.g., sucrose), sugar alcohols suitable for use with diabetic patients (e.g., sorbitol or mannitol), or other sweeteners (e.g., the sweeteners described herein) may be used. The candy base can be very soft and dissolve quickly, or it can be hard and dissolve more slowly. Various shapes will have advantages in different situations.

A candy mass composition comprising an effective amount of the glycan polymer can be administered orally to a subject in need so that an effective amount of the glycan polymer is released into the subject's mouth as the candy mass It dissolves and is ingested. A subject in need includes a human adult or child.

The dosage forms described herein may also take the form of pharmaceutical particles manufactured by a variety of methods, including, but not limited to, high pressure homogenization, wet or dry ball milling, or particle precipitation. small (e.g. NanoSpray from nGimat). Other useful methods for preparing a suitable powder formulation are the preparation of a solution of active ingredients and excipients, followed by precipitation, filtration and spraying, or followed by removal of the solvent by freeze drying, followed by spraying the powder to the size of desired particle. In one embodiment, the pharmaceutical particles have a final size of 3-1000 micrometers, such as at most 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80 , 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 micrometers. In another embodiment, the pharmaceutical particles have a final size of 10-500 micrometers. In another embodiment, the pharmaceutical particles have a final size of 50-600 micrometers. In another embodiment, the pharmaceutical particles have a final size of 100-800 micrometers.

In another aspect, the disclosure provides a method of preparing a unit dosage form described herein, comprising providing a glycan polymer (e.g., a glycan polymer described herein); formulate the glycan polymer into a unit dosage form (e.g., a unit dosage form described herein), package the unit dosage form, label the packaged unit dosage form, and/or sell or offer for sale the packaged and labeled unit dosage form.

The unit dosage forms described herein may also be processed. In one embodiment, the processing comprises one or more of: processing the dosage form into a pharmaceutical composition, e.g., formulating, combining with a second component, e.g., an excipient or buffer; divide into smaller or larger aliquots; providing in a container, for example, a gas or liquid tight container; package; associate with a tag; send or move to a different location. In one embodiment, processing comprises one or more of: sorting, sorting, accepting or discarding, releasing or holding, processing into a pharmaceutical composition, shipping, moving to a different location, formulating, labeling, packaging, releasing to commerce or selling or offer for sale, depending on whether the predetermined threshold is met. In some embodiments, the processed dosage forms comprise a glycan polymer described herein.

In some embodiments, the processing comprises one or more of: processing the dosage form into a pharmaceutical composition, e.g., formulating, combining with a second component, e.g., an excipient or buffer; divide into smaller or larger aliquots; providing in a container, for example, a gas or liquid tight container; package; associate with a tag; send or move to a different location. In one embodiment, processing comprises one or more of: sorting, sorting, accepting or discarding, releasing or holding, processing into a pharmaceutical composition, shipping, moving to a different location, formulating, labeling, packaging, releasing to commerce or selling or offer for sale, depending on the determination.

In another embodiment, an oral dosage form is provided comprising a glycan polymer preparation, wherein the oral dosage form is a syrup. The syrup may comprise approximately 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% , 75%, 80% or 85% solid. The syrup may comprise approximately 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% liquid, for example, water. The solid may comprise a glycan polymer preparation. The solid may be, for example, about 1-96%, 10-96%, 20-96%, 30-96%, 40-96%, 50-96%, 60-96%, 70-96%, 80-96% or 90-96% glycan polymer preparation. In another embodiment, a glycan polymer preparation is formulated as a viscous fluid.

In one embodiment, the composition comprises a foaming component, a neutralizing component, or a water-insoluble dietary fiber. A foaming component may be at least one member selected from the group consisting of sodium hydrogen carbonate, sodium carbonate and calcium carbonate. In one embodiment, a neutralizing component may be at least one member selected from the group consisting of citric acid, L-tartaric acid, fumaric acid, L-ascorbic acid, DL-malic acid, acetic acid, lactic acid and anhydrous citric acid. In one embodiment, a water-insoluble dietary fiber may be at least one member selected from the group consisting of crystalline cellulose, wheat bran, oat bran, corn fiber, soybean fiber, and beet fiber. The formulation may contain a sucrose fatty acid ester, powdered sugar, powdered fruit juice and/or flavoring material.

In some embodiments, the dosage forms are formulated to release the pharmaceutical compositions comprising glycan polymer preparations into specific region(s) of the gastrointestinal tract, such as the small intestine or the large intestine. In some embodiments, the dosage forms are formulated to release the pharmaceutical compositions comprising glycan polymer preparations in specific region(s) of the gastrointestinal tract, such as the cecum, ascending colon, transverse colon, descending colon. , the sigmoid colon and/or rectum.

In some embodiments, the dosage form for the glycan polymer preparations described herein is an enzyme-sensitive delivery system. For example, trypsin-sensitive polymers can be prepared using hydrogels that are cross-linked by peptides that are degraded by trypsin. Trypsin is active in the small intestine. Trypsin-sensitive delivery systems can be used to direct the delivery of glycan polymer preparations to the small intestine. In another example, enzyme-digestible hydrogels consisting of poly(vinylpyrrolidone) cross-linked with albumin are degraded in the presence of pepsin.

In some embodiments, the dosage form for the glycan polymer preparations described herein is a delivery device that allows for prolonged retention at a specific site in the gastrointestinal tract. For example, a gastroretentive delivery system allows for sustained release of glycan polymer preparations to the stomach. Gastroretentive administration can be used for glycan polymer preparations that modulate bacteria in the stomach or upper small intestine.

In some embodiments, the dosage form for the glycan polymer preparations described herein is a mucoadhesive delivery system that adheres to the mucosal surfaces of the stomach. Typically, they are composed of polymers with numerous hydrogen bonding groups, for example, cross-linked poly(acrylic acids), sodium carboxymethyl cellulose, sodium alginate, carrageenan, Carbopol 934P or thiolated polycarbophil.

In some embodiments, the dosage form for the glycan polymer preparations described herein is an expanding delivery system that rapidly increases in size in the stomach, slowing its passage through the pylorus. Such systems include systems that deploy in the stomach. For example, geometric shapes such as tetrahedra, rings, disks, etc. can be packaged. in a gelatin capsule. When the capsule dissolves, the shape unfolds. The systems may be composed of one or more erodible polymers (eg, hydroxypropyl cellulose), one or more non-erodible polymers (eg, polyolefins, polyamides, polyurethanes). The glycan polymer can then be dispersed within the polymer matrix. Retention times can be finely tuned by mixing polymers. Alternatively, devices made of elastic polymers that are stable in the acidic pH of the stomach but dissolve in neutral/alkaline conditions along the GI tract can be used. Such polymer formulations can prevent intestinal obstruction when the device exits the stomach. Supramolecular polymeric gels cross-linked by hydrogen bonds between carboxyl groups can also be used, for example, composed of poly(acryloyl-6-aminocaproic acid) (PA6ACA) and poly(ethyl methacrylic acid-co-acrylate) (EUDRAGIT L 100-55 ). Other systems include swellable excipients, such as collagen sponges. For example, a hydrogel matrix (e.g., a swellable core: polyvinylpyrrolidone XL, Carbopol 934P, calcium carbonate) swells 2-50 times in the stomach. Superporous hydrogel composites swell to hundreds of times their original volume within a few minutes. Some systems take advantage of gas generation to achieve expansion, for example, expandable systems that generate carbon dioxide and are surrounded by a hydrophilic membrane.

In some embodiments, the dosage form for the glycan polymer preparations described herein is a controlled density delivery system. These systems are designed to either float or sink in gastric fluids, which delays their emptying from the stomach. For example, high-density systems allow the device to settle to the bottom of the stomach, below the pylorus, thus preventing emptying of the stomach. Other systems are floating/low density systems. Such devices, for example, may comprise air trapped in hollow chambers or may incorporate low density materials such as greases, oils or powdered foam. Low density can be achieved through swelling, for example, capsules containing hydrocolloids dissolve upon contact with gastric fluid and the hydrocolloids swell to form a mucous body. Alternative polymers include: chitosans, sodium alginate and glycerol monooleate matrix. Low density can be achieved through gas generation. For example, tablets loaded with carbonate and optionally citric acid generate carbon dioxide after contact with acidic aqueous media. The carbon dioxide generated is trapped within the gelling hydrocolloid causing the system to float. Hydrocolloids include hydroxypropylmethylcellulose and Carbopol 934P.

In some embodiments, the dosage form for the glycan polymer preparations described herein employs a design to retain a device in the small or large intestine. The location-specific nature of the device is provided by a specific activation method, e.g. pH, enzymes, etc. These include systems designed for mucoadhesion and also microneedling pills. Microneedle pills comprise a microneedle-enriched drug reservoir that is encapsulated in a pH-sensitive coating. When the pill reaches the desired location in the GI tract and the coating dissolves, the microneedles allow the pill to adhere to the lining of the GI tract. In other embodiments, the microneedle pills comprise a capsule consisting of two chemical compartments filled with citric acid and sodium bicarbonate, respectively. As the pill dissolves in the digestive system, the barriers between the two substances erode, allowing them to mix and create a chemical reaction that pushes the saccharide microneedles through the outer layer of the capsule and into the interior lining. of the small intestine. Saccharide needles may be filled with drugs that are delivered to nearby blood vessels as the saccharide is absorbed.

In some embodiments, the dosage form for the glycan polymer preparations described herein employs a pH-sensitive polymer coating. For example, pH-dependent polymers (biphasic or triphasic) may be insoluble at low pH levels (e.g., stomach acid resistance, pH 1-2) and become increasingly soluble as the pH is raised. pH, for example, up to about 5.5-6.2 in the duodenum, up to about pH 5.7 in the ascending colon, up to about pH 6.4 in the cecum, up to about pH 6.6 in the transverse colon, up to approximately pH 7.0 in the descending colon, up to approximately 7.2 - 7.5 in the ileum or up to approximately pH 7.5 in the distal small intestine. In one example, TARGIT™ technology can be used for site-specific delivery of glycan polymer preparations to the gastrointestinal (GI) tract. The system uses pH-sensitive coatings on injection-molded starch capsules to target the terminal ileum and colon. In some embodiments, the dosage form for the glycan polymer preparations described herein is a delayed release system or a time-controlled release system. Such systems typically employ enteric coatings that can be combined with pH-sensitive and extended-release functions. For example, ETP (enteric coated time release pressure coated) tablets can be used which are composed of three components: a central tablet containing glycan polymer (rapid release function), a swellable and coated hydrophobic polymer layer pressure (e.g. hydroxypropyl cellulose (HPC) layer), and a time release function. The duration of the lag phase can be controlled by either the weight or the composition of the polymer layer and an enteric coating layer (acid resistance function).

In some embodiments, the dosage form for the glycan polymer preparations described herein employs Eudragit® enteric coatings of tablets and capsules. Other suitable synthetic polymers include: shellac coatings, ethylcellulose, cellulose acetate phthalate, hydroxypropylmethylcellulose, poly(vinyl acetate phthalate) and poly(glutamic acid), such as poly-y-glutamic acid (y-PGA). These coatings combine both mucoadhesive and pH-dependent release strategies. To improve targeted delivery to the colon, Eudragits® are methacrylic copolymers with variable side group compositions that alter the pH at which they are soluble. For example, for systems coated with Eudragit®, no significant drug release occurs in the stomach (e.g., at pH 1.4) or small intestine (e.g., at pH 6.3), while there may be A significant release of drug was observed at pH 7.8 in the ileocecal region.

In some embodiments, the dosage form for the glycan polymer preparations described herein is a microbially activated system, such as a polysaccharide-based delivery system. Polysaccharide-based delivery systems contain biodegradable and mucoadhesive polymer coatings, including chitosan and pectin coatings. Other suitable natural polymers include, for example, guar gum, inulin, cyclodextrin, dextran, amylase, chondrotin sulfate and locust bean gum. These delivery systems can be used to target the glycan polymer to the small intestine. Coatings with naturally occurring polysaccharides such as guar gum, xanthan gum, chitosan, alginates, etc. They are degraded by the intestinal microbiota of the colon, for example, enzymes such as xylosidase, arabinosidase, galactosidase, etc. For example, you can use CODES™ technology to deliver glycan polymer preparations. This system combines the polysaccharide coating with a pH sensitive coating. In some embodiments, the system consists of a central tablet coated with three layers of polymeric coatings: the outer coating is composed of Eudragit L. This coating dissolves in the duodenum and exposes the next coating. The next coating is made up of Eudragit E. This layer allows the release of the lactulose present in the inner core. Lactulose is metabolized into short chain fatty acids which lower the surrounding pH where the Eudragit E layer dissolves. Dissolution of Eudragit E results in exposure of the glycan polymer. The bacteria present in the colon are responsible for the degradation of the polysaccharides that are released from the tablet core. The degradation of polysaccharides can result in the formation of organic acids that reduce the pH of the contents surrounding the tablet.

In some embodiments, the dosage form for the glycan polymer preparations described herein is a pressure-controlled delivery system. The system takes advantage of the fact that higher pressures are found in the colon than in the small intestine. For example, for ethylcellulose systems that are insoluble in water, the release of glycan polymers occurs after disintegration of a water-insoluble polymer capsule as a result of pressure in the lumen of the colon. The release profile can be adjusted by varying the thickness of the ethyl cellulose, the size of the capsule and/or the density of the capsule.

In some embodiments, the dosage form for the glycan polymer preparations described herein is a pulsatile delivery system directed to the colon. For example, the system may be a pulsating capsule system. The capsule used comprises a plug that is placed in the capsule and that controls the release of the glycan polymer. A swellable hydrogel (e.g., hydroxypropylmethylcellulose (HPMC), poly(methyl methacrylate), or poly(vinyl acetate)) seals the drug contents. When the capsule comes into contact with a fluid, the plug dislodges from the capsule and the glycan polymer is released. The release profile can be controlled by varying the length and/or the point of intersection of the plug with the capsule body. Another system is a port system. The body of the capsule is enclosed in a semipermeable membrane. The insoluble plug consists of an osmotically active agent and the glycan polymer. When the capsule comes into contact with a fluid, the semipermeable membrane allows fluid inflow, increasing the pressure in the capsule body. This leads to the expulsion of the plug and the release of the glycan polymer.

In some embodiments, the dosage form for the glycan polymer preparations described herein is an osmotically controlled colon-targeted delivery system. An exemplary system, OROS-CT, consists of osmotic units (up to 5 or 6 push-pull units) encapsulated in a hard gelatin capsule. Push-pull units have two layers with an outer impermeable enteric membrane and an inner semipermeable membrane. The inner core part of the push-pull consists of the drug layer and the push layer. The glycan polymer is released through the semipermeable membrane. The capsule body enclosing the push-pull units dissolves immediately after administration. In the GI tract, the impermeable enteric membrane prevents water absorption. The enteric coating dissolves in the small intestine (higher pH, >7), water enters the unit through the semipermeable membrane, causing the thrust layer to swell and expel the glycan polymer.

In some embodiments, the dosage form for the glycan polymer preparations described herein is a “smart pill” that can be used to release the glycan polymer just before reaching the ileocecal valve.

In some embodiments, the dosage form for the glycan polymer preparations described herein is a formulation administered rectally. For example, enemas introduce a glycan polymer preparation in liquid formulation into the rectum. The volume administered is normally less than 10 ml. Suppositories introduce a glycan polymer preparation into the rectum. Suppositories are solid dosage forms that melt or dissolve when inserted into the rectum, releasing the glycan polymers. Typical excipients for suppository formulations include cocoa butter, polyethylene glycols and agar.

Kits

The following kits are for illustrative purposes.

Kits are also included. For example, a kit may comprise unit dosage forms of the glycan polymer preparation and a package insert containing instructions for use of the glycan polymer in the treatment of a gastrointestinal disorder or condition. The kits include a glycan polymer preparation in a container suitable for use by a subject (e.g., a human subject) in need. Any of the compositions described herein can be packaged in the form of a kit. A kit may contain an amount of a glycan polymer preparation (optionally further comprising a prebiotic substance, a probiotic bacteria and/or a second therapeutic agent) sufficient for a complete treatment cycle, or for a portion of a treatment cycle. . Doses of a glycan polymer preparation may be packaged individually, or the glycan polymer preparation may be provided in bulk, or combinations thereof. Thus, in one embodiment, a kit provides, in a suitable container, individual doses of a glycan polymer preparation corresponding to dosing points in a treatment regimen, where the doses are packaged in one or more containers.

In one embodiment, the glycan polymer preparation may be provided in bulk in a single container, or in two, three, four, five or more than five containers. For example, each container may contain enough glycan polymer preparation for a particular week of a month-long treatment program. If more than one bulk container is provided, the bulk containers may be suitably packaged together to provide sufficient glycan polymer preparation for all or part of a treatment period. The container or containers may be labeled with a label indicating information useful to the subject in need or to the physician performing the treatment protocol, such as, for example, dosing schedules.

The glycan polymer preparation may be packaged with other suitable substances, such as probiotic bacteria, prebiotic substances or other substances, as described herein. The other substance or substances may be packaged separately from the glycan polymer preparation, or mixed with the glycan polymer preparation, or combinations thereof. Therefore, in one embodiment, the kits include a dosage form containing all components intended for use in a treatment course or a part of a treatment course, for example, a glycan polymer preparation and optionally buffers, excipients , etc., a probiotic, prebiotic or a polymer agent. In one embodiment, a glycan polymer preparation is packaged in a container or set of containers, and additional components, such as probiotic bacteria, prebiotics, and therapeutic agents, are packaged separately from the glycan polymer preparation.

The kits may also include written materials, such as instructions, expected results, testimonials, explanations, warnings, clinical data, information for health professionals and the like. In one embodiment, the kits contain a label or other information indicating that the kit is for use only under the direction of a healthcare professional. The container may also include spoons, syringes, bottles, cups, applicators or other measuring or serving devices.

Identification of bacterial constituents

The following aspects and embodiments for identifying bacterial constituents are for illustrative purposes.

In some embodiments, the glycan polymer preparations described herein are administered to a subject (e.g., a human subject) to increase the growth of beneficial bacteria, decrease the growth of pathogens, and/or modulate a (microbial) metabolite. (such as, for example, SCFA, ammonia, TMA/TMAO, bile acids, LPS) in the GI tract. In some embodiments, the glycan polymer shifts the microbial community toward a healthy state. Microbial changes that occur in the GI tract can be analyzed using any of several methods known in the art and described herein. As a quantitative method to determine whether a glycan polymer preparation results in a change in the population of bacteria in the GI tract, quantitative PCR (qPCR) can be performed. Genomic DNA can be extracted from samples using commercially available kits, such as the Mo Bio Powersoil®-htp 96 Well Soil DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA), the Mo Bio Powersoil® DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA), or the QIAamp DNA Stool Mini kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions, or by other conventional methods known to those skilled in the art.

In some embodiments, qPCR can be performed using HotMasterMix (5PRIME, Gaithersburg, MD) and primers specific for certain bacteria (e.g., beneficial or desired) and can be performed in a MicroAmp® Fast Optical barcoded 96-well reaction plate ( 0.1 ml) (Life Technologies, Grand Island, NY) and performed in a BioRad C1000™ thermal cycler equipped with a CFX96™ real-time system (BioRad, Hercules, CA), with fluorescent readings from the FAM and ROX channels. The Cq value for each well in the FAM channel is determined by CFX Manager™ software version 2.1. The log(cfu/ml) of each experimental sample is calculated by entering the Cq value of a given sample into a linear regression model generated from the standard curve by comparing the Cq values of the wells of the standard curve with the log10 (cfu/ml) known from those samples. One skilled in the art may employ alternative qPCR modes. In some embodiments, microbial constituents are identified by characterizing the DNA sequence of the microbial 16S small subunit ribosomal RNA gene (16S rRNA gene). The 16S rRNA gene is approximately 1,500 nucleotides in length and is generally highly conserved across organisms, but contains specific variable and hypervariable regions (V1-V9) that harbor sufficient nucleotide diversity to differentiate taxa at the species and strain level. of most organisms. These regions in bacteria are defined by nucleotides 69-99, 137-242, 433-497, 576-682, 822-879, 986 1043, 1117-1173, 1243-1294 and 1435-1465 respectively using numbering based on the nomenclature system of E. coli. (see, for example, Brosiuset al., Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli, PNAS 75(10):4801-4805 (1978).)).

The composition of a microbial community can be deduced by sequencing the entire 16S rRNA gene, or at least one of the V1, V2, V3, V4, V5, V6, V7, V8 and V9 regions of this gene or by sequencing any combination of variable regions of this gene (for example, V1-3 or V3-5). In one embodiment, the V1, V2 and V3 regions are used to characterize a microbiota. In another embodiment, the V3, V4 and V5 regions are used to characterize a microbiota. In another embodiment, the V4 region is used to characterize a microbiota.

Sequences that are at least 97% identical to each other are grouped into Operational Taxonomic Units (OTUs). OTUs containing sequences with 97% similarity roughly correspond to species-level taxa. At least one representative sequence is chosen from each OTU and used to obtain a taxonomic assignment for an OTU by comparison with a reference database of highly curated 16S rRNA gene sequences (such as the Greengenes or SILVA databases). . The relationship between OTUs in a microbial community could be inferred by constructing a phylogenetic tree from representative sequences of each OTU.

Using known techniques, in order to determine the complete 16S sequence or the sequence of any variable region of the 16S sequence, genomic DNA is extracted from a bacterial sample, the 16S rRNA (complete region or specific variable regions) is amplified. Using polymerase chain reaction (PCR), PCR products are cleaned and nucleotide sequences are delineated to determine the genetic composition of the 16S rRNA gene or a variable region of the gene. If full 16S sequencing is performed, the sequencing method used may be, but is not limited to, Sanger sequencing. If one or more variable regions, such as the V4 region, are used, sequencing may be performed, but not limited to, using the Sanger method or using a next generation sequencing method, such as an Illumina method. Primers designed to hybridize to conserved regions of 16S rRNA genes (e.g., primers 515F and 805R for amplification of the V4 region) could contain unique barcode sequences to allow characterization of multiple microbial communities simultaneously.

As another method to identify microbial composition, there is the characterization of genes or nucleotide markers, in particular highly conserved genes (e.g., “housekeeping” genes), or a combination thereof, or an indiscriminate sequence of the entire genome ( WGS). Using defined methods, DNA extracted from a bacterial sample will have specific genomic regions amplified using PCR and will be sequenced to determine the nucleotide sequence of the amplified products. In the WGS method, the extracted DNA will be fragmented into pieces of various lengths (from 300 to approximately 40,000 nucleotides) and sequenced directly without amplification. Sequence data may be generated using any sequencing technology including, but not limited to, Sanger, Illumina, 454 Life Sciences, Ion Torrent, ABI, Pacific Biosciences and/or Oxford Nanopore.

In addition to the 16S rRNA gene, a selected set of genes known to be marker genes for a given species or taxonomic group are analyzed to assess the composition of a microbial community. Alternatively, these genes are analyzed using a PCR-based detection strategy. For example, various strains of colipathogenic Escherichia are distinguished using genes that encode heat-labile (LTI, Ltha and LTIIb) and heat-stable toxins (STI and STII), verotoxin types 1, 2 and 2e (VT1, VT2 and VT2e, respectively), necrotizing factors cytotoxic (CNF1 and CNF2), attachment and erasure mechanisms (eaeA), enteroaggregative mechanisms (Eagg) and enteroinvasive mechanisms (Einv). The optimal genes to be used to determine the taxonomic composition of a microbial community using marker genes are familiar to one of ordinary skill in the art of sequence-based taxonomic identification.

Sequencing libraries for microbial whole genome sequencing (WGS) can be prepared from bacterial genomic DNA. For genomic DNA that has been isolated from a human sample or a laboratory animal, the DNA can optionally be enriched with bacterial DNA using commercially available kits, for example, the NEBNext Microbiome DNA Enrichment Kit (New England Biolabs, Ipswich , MA) or other enrichment kit. Sequencing libraries can also be prepared from genomic DNA using commercially available kits, such as the Nextera Mate-Pair Sample Prep Kit, TruSeq DNA PCR-Free, or TruSeq Nano DNA, or the Nextera XT Sample Prep Kit (Illumina , San Diego, United States, CA) according to the manufacturer's instructions. Alternatively, libraries can be prepared using other kits compatible with the Illumina sequencing platform, such as the NEBNext DNA Library Construction Kit (New England Biolabs, Ipswich, MA). Libraries can then be sequenced using conventional sequencing technology including, but not limited to, a MiSeq, HiSeq, or NextSeq sequencer (Illumina, San Diego, CA).

Alternatively, a library of indiscriminate whole-genome fragments can be prepared using conventional methods in the art. For example, the indiscriminate fragment library could be constructed using the GS FLX Titanium Rapid Library Prep Kit (454 Life Sciences, Branford, CT), amplified using a GS FLX Titanium emPCR Kit (454 Life Sciences, Branford, CT), and sequenced. following conventional 454 pyrosequencing protocols on a 454 sequencer (454 Life Sciences, Branford, CT).

Bacterial RNA can be isolated from microbial cultures or samples containing bacteria using commercially available kits, such as the RiboPure Bacterial RNA Purification Kit (Life Technologies, Carlsbad, CA). Another method for bacterial RNA isolation may involve enrichment of mRNA in purified bacterial RNA samples through tRNA knockdown. Alternatively, RNA can be converted to cDNA, which is used to generate sequencing libraries using conventional methods such as the Nextera XT sample preparation kit (Illumina, San Diego, CA).

Nucleic acid sequences are analyzed to define taxonomic assignments using sequence similarity and phylogenetic placement methods or a combination of the two strategies. A similar approach is used to annotate protein names, protein functions, transcription factor names, and any other classification scheme for nucleic acid sequences. Methods based on sequence similarity include BLAST, BLASTx, tBLASTn, tBLASTx, RDP-classifier, DNAclust, RapSearch2, DIAMOND, USEARCH and various implementations of these algorithms such as QIIME or Mothur. These methods map a sequence read to a reference database and select the best match. Common databases include KEGG, MetaCyc, NCBI non-redundant database, Greengenes, RDP, and Silva for taxonomic assignments. For functional assignments, reads are mapped to various functional databases, such as COG, KEGG, BioCyc, MetaCyc, and the carbohydrate-active enzymes (CAZy) database. Microbial clades are assigned using software included MetaPhlAn.

Proteomic analysis of microbial populations.

Preparations of glycan polymers can be selected based on their ability to increase the expression of microbial proteins associated with healthy states or to decrease the expression of microbial proteins associated with disease states. Proteomic analysis of microbial populations can be performed following protocols known to one skilled in the art (for example, Cordwell, Exploring and exploiting bacterial proteomes, Methods in Molecular Biology, 2004, 266:115). To identify differentially expressed proteins (for example, to identify changes in protein expression following treatment of microbial populations with glycan polymers), proteomic analysis can be performed as described, for example, in Justeet al. (Bacterial protein signals are associated with Crohn's disease, Gut, 2014, 63:1566). For example, the protein is isolated from the microbial lysates of two samples (e.g., an untreated microbial population and a population that has been treated with glycan polymers). Each protein sample is labeled (e.g., with a fluorescent dye, e.g., Cy3 or Cy5 minimal dye CyDye DIGE Fluor, GE Healthcare) and analyzed by two-dimensional differential gel electrophoresis (2D-DIGE). The gels are stained and protein spots identified as significantly different between the two samples are cut, digested and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). X!TandemPipeline (http://pappso.inra.fr/bioinfo/xtandempipeline/) can be used to identify differentially expressed proteins.

Preparations of glycan polymers can also be selected for administration to a human subject based on their effect on the presence of microbial products. For example, glycan polymer preparations may be selected for their ability to induce or promote the growth of bacteria that produce short chain fatty acids such as propionate (propionic acid), acetate and/or butyrate (butyric acid). Similarly, glycan polymer preparations can be selected for their ability to induce or promote the growth of lactic acid-producing bacteria, which can modulate the growth of other bacteria by producing an acidic environment and are also used by butyrate-producing taxa. Such an assay can also be used to pair probiotic bacteria with glycan polymers so that the glycan polymer is a substrate for the production of the desired fermentation products. In some embodiments, glycan polymers may also be selected for administration to a human subject based on their effect on bacterial taxa that do not produce an unwanted metabolite, such as, for example, ammonia, a uremic solute, TMA, and the like. In some embodiments, glycan polymers increase the growth of bacterial taxa that do not produce an unwanted metabolite, thereby successfully competing with bacterial taxa that produce the unwanted metabolite (e.g., for space and nutrients). By shifting the balance from non-producers to producers in favor of non-producers, the overall level of the unwanted metabolite can be reduced. In some embodiments, the balance between SCFA-producing and non-SCFA-producing taxa shifts toward SCFA producers to increase the level of SCFA (e.g., butyrate, acetate, propionate) production. In some embodiments, the balance between ammonia-producing taxa and non-producing taxa is shifted toward non-producing taxa (e.g., urease-negative bacterial taxa) to decrease the level of ammonia production. In some embodiments, the balance of TMA producers to non-producing taxa shifts toward non-producers to decrease the level of TMA production. Metabolites that are present in new or used culture media or in biological samples collected from humans can be determined using methods described herein. Unbiased methods that can be used to determine the relative concentration of metabolites in a sample and are known to a person skilled in the art, such as gas or liquid chromatography combined with mass spectrometry or 1H-NMR. These measurements can be validated by running metabolite standards through the same analytical systems.

In the case of gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS) analysis, polar metabolites and fatty acids could be extracted using single-phase or two-phase systems of organic solvents and a aqueous sample and derivatized (Fendtet al.,Reductive glutamine metabolism is a function of the a-ketoglutarate to citrate ratio in cells, Nat Commun, 2013, 4:2236; Fendtet al.,Metformin decreases glucose oxidation and increases the dependency of prostate cancer cells on reductive glutamine metabolism, Cancer Res, 2013, 73:4429; Metalloet al., Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia, Nature, 2011, 481:380). An exemplary protocol for the derivatization of polar metabolites involves the formation of methoxime-tBDMS derivatives through incubation of the metabolites with 2% methoxylamine hydrochloride in pyridine, followed by the addition of N-tert-butyldimethylsilyl- N-methyltrifluoroacetamide (MTBSTFA) with 1% tert-butyldimethylchlorosilane (t-BDMCS). Non-polar fractions, including triacylglycerides and phospholipids, can be saponified to give free fatty acids and esterified to form fatty acid methyl esters, for example, either by incubation with 2% H2SO4 in methanol or using methyl-8 reagent ( Thermo Scientific). The derivatized samples can then be analyzed by GC-MS using conventional LC-MS methods, for example, a DB-35MS column (30 m x 0.25 mm i.d. x 0.25 |im, Agilent J&W Scientific) installed on a gas chromatograph (GC) interfaced with a mass spectrometer (MS). Mass isotopomer distributions can be determined by integrating ionic fragments of metabolites and corrected for natural stable isotope abundance using conventional algorithms, such as those adapted from Fernández et al. (Fernández et al., Correction of 13C mass isotopomer distributions for natural stable isotope abundance, J Mass Spectrom, 1996, 31:255). For liquid chromatography-mass spectrometry (LC-MS), polar metabolites can be analyzed using a conventional benchtop LC-MS/MS equipped with a column, such as a SeQuant ZIC-pHILIC polymeric column (2, 1 * 150mm; EMD Millipore). Exemplary mobile phases used for separation could include buffers and organic solvents adjusted to a specific pH value.

In combination or alternatively, extracted samples can be analyzed by 1H Nuclear Magnetic Resonance (1H-NMR). Samples can be combined with isotopically enriched solvents such as D2O, optionally in the presence of a buffered solution (e.g. Na2HPO4, NaH2PO4 in D2O, pH 7.4). Samples can also be supplemented with a reference standard for calibration and chemical shift determination (e.g. 5 mM 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt (DSS-d6, Isotec, USA .)). Prior to analysis, the solution can be filtered or centrifuged to remove any sediment or precipitate and then transferred to an NMR tube or container suitable for analysis (e.g., a 5 mm rMn tube). 1H-NMR spectra can be acquired on a conventional NMR spectrometer, such as a Bruker Avance II 500 (500 MHz) spectrometer (Bruker, DE), equipped with a 5 mm QXI-Z C/N/P probe head. ) and analyzed with spectral integration software (such as Chenomx NMR Suite 7.1; Chenomx Inc., Edmonton, AB). (Duarteet al.,1H-NMR protocol for exometabolome analysis of cultured mammalian cells, Methods Mol Biol, 2014:237-47). Alternatively, 1H-NMR can be performed following other published protocols known in the art (Chassainget al., Lack of soluble fiber drives diet-induced adiposity in mice, Am J Physiol Gastrointest Liver Physiol, 2015; Baiet al., Comparison of Storage Conditions for Human Vaginal Microbiome Studies, PLoS ONE, 2012:e36934).

Administration

In some embodiments, a glycan polymer is administered to a subject (e.g., a human subject) in need thereof via enteral administration. In some embodiments, a glycan polymer is administered to a subject in need thereof by oral, nasal, gastric, or rectal administration. In some embodiments, a glycan polymer is administered to a subject in need thereof via tube feeding.

In some embodiments, a glycan polymer is administered to a subject in need thereof immediately after one or more drug treatments have ended (e.g., 1 hour, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, or 4 weeks after ending antibiotic treatment). During a course of drug treatment, the glycan polymer preparation may be provided prior to the start of drug treatment (e.g., 1, 2, 3, 4, 5, 6, 7 days before); the day of initiation of drug treatment; or shortly after antibiotic treatment, for example, 1, 2, 3, 4, 5, 6, 7 or more days after treatment, and optionally may be provided only initially (for example, for a short period) or throughout the duration of drug treatment, and may even be continued for a desired period after the drug treatment period has ended (for example, for 1 to 7 days, 1 to 14 days, or 1 to 21 days after). In some embodiments, administration of the glycan polymer preparation is initiated or continued when one or more adverse effects (e.g., digestive abnormalities or pathogen growth) occur and/or are diagnosed in conjunction with drug treatment. In some embodiments, the treatment agent that causes dysbiosis is not a drug but rather a radiation treatment or surgery and the glycan polymer preparation may also be administered as described herein.

In some embodiments, the total number and duration of treatment periods are based on a subject's response to treatment. For example, an individual may experience a reduction in symptoms after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days of treatment with a polymer preparation of glycan. In another example, an individual may experience a reduction in symptoms after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months of treatment with a glycan polymer preparation. Therefore, the duration of treatment is determined by the response of an individual subject to a glycan polymer preparation and the occurrence of relief of one or more symptoms. Therefore, a subject may experience symptoms with a given dose of a glycan polymer preparation and may require the subject to remain at that dose, or at a lower dose, until the symptoms resolve. Therefore, in one embodiment, the duration of treatment is not determined from the beginning, but continues until the maximum dose of a glycan polymer preparation per day is reached, or until the desired level of reduction of the symptoms. In one embodiment, the treatment is continuous.

In one embodiment, a subject (e.g., a human subject) may be administered a dose for the first treatment period during a treatment regimen and a second dose during a second treatment period. For example, a subject may be administered one dose of glycan polymer preparation over a period of one week and a second dose over a subsequent period of one week.

A subject may self-administer a glycan polymer preparation and the glycan polymer preparation is supplied or recommended (or prescribed) by a healthcare professional, e.g., a physician or other qualified healthcare professional, and test results are optionally monitored ( for example, obtained for biomarkers from samples taken from the subject) and/or health changes and treatment endpoints by a healthcare professional. In some embodiments, the glycan polymer preparation is administered by a healthcare professional.

In one embodiment, a subject in need may undergo repeated courses of treatment with a glycan polymer preparation. The course of treatment may be repeated when symptoms reappear or increase to an unwanted level. Alternatively, the treatment course may be repeated at regular or predetermined intervals. Thus, the treatment can be repeated after approximately one month, two months, three months, four months, six months, eight months, ten months, one year, 18 months, two years, three years, four years, five years or more. five years, or any combination thereof (for example, treatment may be repeated after one year and then every two to five years). Treatment may be repeated in the same manner (e.g., duration, dosage, timing of dosing, additional substances, etc.) as used in the first treatment or may be modified. For example, the duration of treatment can be shortened or lengthened, the dose can be increased or decreased.

In some embodiments, the pharmaceutical composition is administered one, two or three times a day. In some embodiments, the pharmaceutical composition is administered twice daily. In some embodiments, the pharmaceutical composition is administered each day for a predetermined number of days (the treatment period). In some embodiments, the treatment period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 35, 42, 49, 56, 63, 70, 100, 200, 300 or 365 days. In some embodiments, the treatment period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. In some embodiments, the treatment period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 years or for life.

In one embodiment, the total duration of treatment periods for a gastrointestinal disease, disorder or condition may be approximately one day to 10 years, one day to 1 year, 1 day to 6 months, 1 day to 3 months. , from 1 day to 1 month, from one day to one week, from one day to five days, from one day to 10 days, from one week to approximately 12 weeks, or from approximately four weeks to approximately ten weeks, or from approximately four weeks to approximately eight weeks, or approximately six weeks. The subject (for example, a human subject) may undergo an appropriate number of treatment periods, such as, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 treatment periods. During a treatment period, the subject takes a glycan polymer preparation described herein, optionally in conjunction with the ingestion of food products containing prebiotics and/or probiotics. In one embodiment, a glycan polymer preparation may also be administered in combination with another substance (such as a probiotic or commensal beneficial bacteria, a prebiotic substance, or a therapeutic agent), as described herein.

In some embodiments, the glycan polymer preparation may also be combined with an antibiotic that alters the normal growth of the gastrointestinal microbiota. Typically, the duration of antibiotic treatments is 1-14 days, or 2-10 days, or 5-7 days. In some embodiments, a glycan polymer is administered to a subject in need thereof immediately after one or more antibiotic treatments have ended (e.g., 1 hour, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, or 4 weeks after finishing antibiotic treatment). During a course of antibiotic treatment, the glycan polymer preparation may be provided at the beginning of the antibiotic treatment; shortly after antibiotic treatment, for example, 1, 2, 3, 4, 5, 6, 7 or more days after treatment; or may be administered upon diagnosis of unwanted pathogen growth.

Treatment methods

Provided herein are methods for treating a subject (e.g., a human subject) having a disease or disorder. In some embodiments, the disease or disorder is associated with an unwanted level of a metabolite, where the metabolite is trimethylamine (TMA) or trimethylamine N-oxide (TMAO). The methods, in some embodiments, include administering to the human subject a glycan polymer preparation in an amount effective to treat the disease or disorder, wherein the disease or disorder is selected from the group consisting of atherosclerosis, cardiovascular disease, cardiovascular risk in HIV, carotid atherosclerosis, chronic heart disease, chronic heart failure, chronic kidney disease, chronic vascular disease, colorectal cancer, coronary heart disease, coronary artery disease (CAD), diabetes (type II), end-stage renal disease, HIV, inflammatory disease of the intestine, ischemic attack, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), obesity, radiation-induced acute intestinal symptoms (RIAIS) and stroke. The method of treating the subject comprises: selecting a glycan polymer preparation based on the knowledge that it modulates the production or level of the metabolite, and administering an amount of the glycan polymer preparation effective to result in a modulation of the level. of the metabolite, thereby treating the disease or disorder, wherein the glycan polymer preparation comprises glucose and at least one alpha-glycosidic linkage, optionally, wherein the alpha-glycosidic linkage is an alpha-1,3 glycosidic linkage , an alpha-1,4 glycosidic bond, or a combination thereof, and where the average degree of polymerization (DP) of the preparation is between DP3-15. In some embodiments, the glycan polymer preparation (e.g., described herein) is beneficial in the treatment of various diseases, disorders or conditions. Such diseases, disorders or conditions may be associated with a dysbiosis of the microbiota. Alterations in the beneficial microbiota can occur due to a variety of factors (e.g., genetic or environmental), including, but not limited to, the use of antibiotics, chemotherapy products, and other drugs or treatments that induce dysbiosis (e.g., radiation therapy ), pathogen infection, pathobiont activity, miscalibrated caloric intake (e.g., high fat, high sugar), miscalibrated (non-digestible) fiber intake (e.g., low or no fiber), host factors ( for example, host genetic alterations) and the like. In some embodiments, the disease, disorder or condition is associated with a dysbiosis of the gastrointestinal microbiota. In some embodiments, by treating the dysbiosis, the disease, disorder or condition is treated. Symptoms that may be associated with gastrointestinal microbiota dysbiosis and/or a gastrointestinal disease, disorder or condition include, but are not limited to, gas, heartburn, upset stomach, bloating, flatulence, diarrhea, abdominal pain, cramps , nausea, and vomiting. Minor GI-related digestive problems also include occasional bloating, diarrhea, constipation, gas, or an upset stomach.

Indications associated with metabolites

The disease or disorder is associated with an unwanted level of a metabolite, where the metabolite is trimethylamine (TMA) or trimethylamine N-oxide (TMAO). Metabolites, such as a short-chain fatty acid (SCFA), a uremic solute, a lipopolysaccharide, or a bile acid, and the bacteria that produce them, have been associated with a range of diseases and are included for illustrative purposes. For example, reduced levels of butyrate-producing bacteria have been reported in Crohn's disease (Takahashiet al., (2016)), and butyrate and propionate levels are reported to be reduced and acetate increased in fecal samples from patients with Crohn's disease (Galeckaet al., (2013)). Butyrate has been reported to decrease the expression of pro-inflammatory cytokines, which may play an important role in inflammatory bowel disease, including Crohn's disease (Ruso I.et al.PLoS One 2012). Other diseases associated with decreased butyrate levels relative to healthy patient populations include ulcerative colitis (Kumariet al.,2013), type 2 diabetes (Qinet al.,2012), atopic dermatitis (Songet al.,2016), colorectal cancer ( Wanget al.,2012) and Parkinson's disease (Keshavarzianet al.,2015). Administration of glycans that support the growth of microbiota positively associated with butyrate production, directly or indirectly, to individuals may increase levels of butyrate in vivo and improve or prevent symptoms of Crohn's disease.

In some embodiments, it may also be beneficial to administer glycans that reduce the production of one or more short chain fatty acids to treat some diseases. Butyrate-producing bacteria are reported to be increased in obese patients compared to healthy individuals (Rosset al.,2015), and butyrate and propionate have been found to be increased in the stool of obese patients relative to healthy patients (Payneet al.,2011); Likewise, high levels of acetate have been associated with obesity (Gaoet al., 2014). The administration of glycans that selectively decrease the microbiota associated directly or indirectly with an increase in butyrate, propionate and/or acetate may therefore be useful to treat or prevent obesity. Other diseases associated with relatively high levels of acetate include malabsorption syndrome (Balaet al., 2006), colorectal cancer (Weiret al., (2013) and Crohn's disease (Galeckaet al., 2013). Administration of glycans to individuals to Selectively decreasing the microbiota associated directly or indirectly with increased acetate may be useful in the treatment or prevention of diseases associated with elevated acetate levels, such as obesity, malabsorption syndrome, colorectal cancer, and Crohn's disease.

In some embodiments, the disease or disorder is associated with a level (e.g., an unwanted level) of a short chain fatty acid, for example, and is selected from acute pouchitis, allergic diseases, AIDS, atherosclerosis, asthma, dermatitis. atopic, autism spectrum disorder, chronic functional constipation, celiac disease, chronic atrophic gastritis, chronic pouchitis, Clostridium difficile associated disease (CDAD), celiac disease, colorectal adenoma, colorectal cancer, Crohn's disease, cystic fibrosis, depression, diabetes (type I), diabetes (type II), diarrhea, eczema, enterostomy, familial Mediterranean fever, food hypersensitivity, graft versus host disease (GvHD), hepatic encephalopathy, hypertension, inflammatory bowel disease, irritable bowel disease, irritable bowel disease -constipation (IBS-C), lung cancer, microscopic colitis, multiple sclerosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), obesity-related asthma, Parkinson's disease (PD), acute intestinal symptoms radiation-induced, shigellosis, short bowel syndrome, intestinal dysfunction associated with spinal cord injury, systemic inflammatory response syndrome, systemic lupus erythematosus and ulcerative colitis. In some embodiments, treatment methods are provided that include modulating SCFA levels to treat the disease or disorder.

In some embodiments, the disease or disorder is associated with a level (e.g., an unwanted level) of a short chain fatty acid, e.g., butyrate, is diarrhea. In some embodiments, the disease or disorder is associated with a level (e.g., an unwanted level) of a short chain fatty acid, e.g., butyrate, or toxicity, e.g., drug toxicity. In some embodiments, treatment methods are provided that include modulating levels of SCFAs, for example, butyrate to treat the disease or disorder, such as symptoms associated with diarrhea, such as, for example, diarrhea caused by drug toxicity.

In some embodiments, the disease or disorder is associated with a level (e.g., an unwanted level) of trimethylamine or trimethylamine N-oxide, for example, and is selected from atherosclerosis, cardiovascular disease, cardiovascular risk in HIV, atherosclerosis of carotid, chronic heart disease, chronic heart failure, chronic kidney disease (CKD), chronic vascular disease, colorectal cancer, coronary heart disease, coronary artery disease (CAD), diabetes (type II), end-stage renal disease, HIV, inflammatory bowel disease intestine, ischemic attack, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), obesity, radiation-induced acute intestinal symptoms (RIAIS) and stroke. In some embodiments, treatment methods are provided that include modulating TMA or TMAO levels to treat the disease or disorder.

In some embodiments, the disease or disorder is associated with a level (e.g., an unwanted level) of ammonia, for example, and is selected from chronic kidney disease, Helicobacter pylori infection, hepatic encephalopathy, and liver cirrhosis with minimal hepatic encephalopathy. (MHE). In one embodiment, the disease or disorder that is associated with a level (e.g., an unwanted level) of ammonia is hepatic encephalopathy (HE). In some embodiments, treatment methods are provided that include modulating ammonia levels.

In some embodiments, the disease or disorder is associated with a level (e.g., an unwanted level) of a bile acid, for example, and is selected from alcoholic liver cirrhosis, atherosclerosis, chronic pouchitis, cirrhosis, colorectal adenoma, cancer. colorectal, colorectal cancer (postcholecystectonic patients), coronary artery disease, Crohn's disease, cystic fibrosis, inflammatory bowel disease, diabetes (type II), liver disease associated with intestinal failure, irritable bowel disease, irritable bowel disease-constipation (IBS -C), malabsorption syndrome, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), obesity, obesity-related asthma, postcholecystectomy, primary biliary cirrhosis, primary sclerosing cholangitis (PSC), progressive familial intrahepatic cholestasis , reflux esophagitis, short bowel syndrome, Steven Johnson syndrome, ulcerative colitis and uncomplicated diverticular disease. In some embodiments, treatment methods are provided that include modulating the levels of a bile acid to treat the disease or disorder.

In some embodiments, the disease or disorder is associated with a level (e.g., an unwanted level) of lipopolysaccharide, for example, and is selected from allergic diseases, atherosclerosis, autism spectrum disorder, autoimmune hepatitis, chronic fatigue syndromes. (CFS), chronic kidney disease. chronic vascular diseases, common variable immunodeficiency (CVID), Crohn's disease, depression, diabetes (type II), hepatic encephalopathy, hepatitis B, hepatitis C, HIV, HIV elite controllers, liver diseases associated with intestinal failure, bowel disease irritability, metabolic syndrome, neonatal necrotizing enterocolitis (NEC), obesity, Parkinson's disease (PD) and ulcerative colitis. In some embodiments, treatment methods are provided that include modulating LPS levels to treat the disease or disorder.

Drug toxicity/digestive abnormalities (including diarrhea)

Provided herein are methods of reducing drug- or treatment-induced symptoms in a human subject by administering a glycan polymer preparation (e.g., as described herein). In one embodiment, the methods include modulating levels of SCFAs, including butyrate. Symptoms induced by drugs or treatments include any digestive abnormalities. Exemplary digestive abnormalities include, but are not limited to, weight gain, constipation, heartburn, upset stomach, gas, bloating, flatulence, diarrhea, abdominal pain, cramps, nausea and vomiting. In some embodiments, the digestive abnormality is diarrhea. The method includes administering to the human subject a pharmaceutical composition comprising a glycan polymer preparation in an amount effective to reduce one or more symptoms induced by a drug or treatment. In one embodiment, the treatment is radiation treatment. In one embodiment, the treatment is a chemotherapy treatment.

In one embodiment, the subject (e.g., a human subject) who is identified as suitable for treatment with a glycan polymer preparation has or is suspected of having drug-induced diarrhea, drug-induced constipation, drug-induced toxicity, drug-induced intolerance (e.g., to metformin, to chemotherapies, such as, e.g., irinotecan (camptosar) and/or 5-fluorouracil), drug-induced microbiome damage, drug-induced microbiome disease, drug-induced gastrointestinal disease drugs, drug-induced enteritis or colitis or similar drug-induced disorder or condition.

In some embodiments, the pharmaceutical composition comprising a glycan polymer preparation is administered before, concomitantly with, or after administration of the drug (or radiation treatment), the administration of which induces symptoms.

Exemplary drugs that are often associated with drug- or treatment-induced symptoms include, but are not limited to, an anticancer drug, an antidiabetic drug, an immunosuppressive drug, an antimicrobial drug, a chemotherapy drug, an antipsychotic, a proton pump inhibitor, tyrosine kinase inhibitors (TKIs, such as dasatinib (Sprycel), erlotinib (Tarceva), gefitinib (Iressa), imatinib (Gleevec), lapatinib (Tykerb), nilotinib (Tasigna), sorafenib (Nexavar ), sunitinib (Sutent), afatinib (Gilotrif), alectinib (Alecensa), axitinib (Inlyta), bortezomib (Velcade), bosutinib (Bosulif), cabozantinib (Cometriq, Cabometyx), carfilzomib (Kyprolis), ceritinib (Zykadia), cobimetinib (Cotellic), crizotinib (Xalkori), dabrafenib (Tafinlar), dasatinib (Sprycel), erlotinib (Tarceva), gefitinib (Iressa), ibrutinib (Imbruvica), idelalisib (Zydelig), imatinib (Gleevec), ixazomib (Ninlaro), lapatinib (Tykerb), lenvatinib (Lenvima), nilotinib (Tasigna), niraparib (Zejula), olaparib (Lynparza), osimertinib (Tagrisso), palbociclib (Ibrance), pazopanib (Votrient), pegaptanib (Macugen), ponatinib (Iclusig), regorafenib (Stivarga), ribociclib (Kisqali), rucaparib (Rubraca), ruxolitinib (Jakafi), sonidegib (Odomzo), sorafenib (Nexavar), sunitinib (Sutent), tofacitinib (Xeljanz), trametinib (Mekinist), vandetanib (Caprelsa), vemurafenib (Zelboraf), vismodegib (Erivedge), and a nonsteroidal anti-inflammatory drug (NSAID). The administration of these drugs is generally associated with dysbiosis which may occur, for example, during the treatment regimen. In some embodiments, the dysbiosis causes or amplifies symptoms induced by the drug or treatment, such as digestive abnormalities, such as diarrhea. In some embodiments, administration of the glycan polymer preparation modulates the microbiome such that symptoms induced by the drug or treatment are reduced (for example, by modulating levels of SCFAs, such as butyrate). In some embodiments, the glycan polymer preparation promotes the growth of commensal bacteria and/or supports the growth of beneficial microbial communities that would be negatively affected or lost in response to drug treatment or that may complement the commensal bacteria that have been seen. negatively affected or lost in response to pharmacological treatment. In some embodiments, the glycan polymer preparation promotes the growth of SCFA-producing bacterial taxa, such as, for example, acetate-, propionate-, or butyrate-producing taxa.

Specific examples of drugs associated with digestive abnormalities whose symptoms can be reduced by administration of the glycan polymer preparation include, but are not limited to, ciprofloxacin, clindamycin, amoxicillin-clavulanate, cefixime, cephalosporins, fluoroquinolones, azithromycin, clarithromycin, erythromycin , tetracycline, azithromycin, irinotecan (camptosar), 5-fluorouracil, leucovorin, oxaliplatin, bortezomib, imatinib, lenalidomide, imbruvica, ipilimumab, pertuzumab, capecitabine, docetaxel, lapatinib, erlotinib, carmustine, etoposide, aracitin, melphalan, cytarabine, daunorubicin, amsacrine, mitoxantrone, olanzapine, ranitidine, famotidine, cimetidine, omeprazole, sucralfate, esomeprazole, naproxen, diclofenac, indomethacin, ibuprofen, ketoprofen, piroxicam, celecoxib, nimesulide, aspirin, metformin, paroxetine, valproic acid or clozapine.

In some embodiments, digestive abnormalities are associated with treatment of the subject (e.g., a human subject) with a chemotherapeutic agent. In one embodiment, the digestive abnormality is diarrhea. In specific embodiments, the chemotherapeutic agent is irinotecan, 5-fluorouracil, leucovorin or combinations thereof. In specific embodiments, the chemotherapeutic agent is oxaliplatin, leucovorin, 5-fluorouracil or combinations thereof (e.g., FOLFIRI regimen). In specific embodiments, the chemotherapeutic agent is bortezomib, imatinib, lenalidomide, imbruvica, ipilimumab, pertuzumab, capecitabine, docetaxel, lapatinib, erlotinib or combinations thereof. In some embodiments, the chemotherapeutic agent is carmustine, etoposide, aracitin, melphalan or combinations thereof. In specific embodiments, the chemotherapeutic agent is cytarabine, daunorubicin, etoposide or combinations thereof. In specific embodiments, the chemotherapeutic agent is amsacrine, cytarabine, etoposide or combinations thereof. In specific embodiments, the chemotherapeutic agent is mitoxantrone, cytarabine or combinations thereof. In some embodiments, digestive abnormalities are associated with treatment of the subject with an antibiotic. In one embodiment, the digestive abnormality is diarrhea. In specific embodiments, the antibiotic is ciprofloxacin, clindamycin, amoxicillin-clavulanate, cefixime, cephalosporins, fluoroquinolones, azithromycin, clarithromycin, erythromycin, tetracycline or azithromycin.

In some embodiments, digestive abnormalities are associated with treatment of the subject with an antipsychotic drug. In one embodiment, the digestive abnormality is weight gain. In one embodiment, the drug is olanzapine.

In some embodiments, digestive abnormalities are associated with treatment of the subject with a proton pump inhibitor drug. In one embodiment, the digestive abnormality is diarrhea. In specific embodiments, the drug is ranitidine, famotidine, cimetidine, omeprazole, sucralfate or esomeprazole.

In some embodiments, digestive abnormalities are associated with treatment of the subject with a non-steroidal anti-inflammatory drug (NSAID). In one embodiment, the digestive abnormality is diarrhea. In specific embodiments, the drug is naproxen, diclofenac, indomethacin, ibuprofen, ketoprofen, piroxicam, celecoxib, nimesulide or aspirin.

In some embodiments, digestive abnormalities are associated with treatment of the subject with metformin, paroxetine, valproic acid, or clozapine.

In one embodiment, reduction of one or more symptoms increases the subject's compliance with the treatment regimen. In one embodiment, reducing one or more symptoms allows the physician to prescribe a higher dose of the drug to be administered. In such embodiments, treatment of the underlying disease is more effective (e.g., greater reduction in symptoms, shorter period to achieve a symptom- or disease-free state, or longer maintenance of a symptom-free or disease-free state). illness, etc.).

Chronic kidney disease (CKD)

In some embodiments, subjects with chronic kidney disease (CKD) may be treated according to the methods provided herein. Subjects with CKD may experience fatigue, difficulty concentrating, poor appetite, difficulty sleeping, nighttime muscle cramps, swollen feet and ankles, rash/itching, nausea, vomiting, metallic taste in the mouth, difficulty breathing, and/or increased urination. Diagnosis of kidney disease, including CKD, is made by testing glomerular filtration rate (GFR), blood levels of urea and creatinine, urine albumin levels, kidney biopsy, ultrasound, and/or CT scan. Patient populations include subjects with CKD caused by diabetic nephropathy; subjects with CKD caused by high blood pressure; subjects with polycystic kidney disease, pyelonephritis or glomerulonephritis; subjects with kidney damage due to long-term use of medications that damage the kidneys; and subjects at risk of developing CKD due to the presence of risk factors such as diabetes, high blood pressure, or family history of kidney disease.

Hepatic encephalopathy (HE)

In some embodiments, subjects with hepatic encephalopathy (HE) may be treated according to the methods provided herein. Hepatic encephalopathy includes multiple adverse neurological symptoms that occur when the liver cannot remove toxic substances such as ammonia from the blood. Subjects with HE may present with confusion, forgetfulness, anxiety or excitement, sudden changes in personality or behavior, changes in sleep patterns, disorientation, sweet or musty breath, difficulty speaking, and/or difficulty controlling functions. motors. The diagnosis of HE is made by liver function tests, serum ammonia levels, EEG, and other blood and neurological tests. Patient populations include subjects with mild HE, severe HE, overt HE, subjects who have previously experienced one or more episodes of HE, and patients who are at risk for HE due to the presence of risk factors such as liver damage.

Inflammatory bowel disease (IBP)/Crohn's disease (CD)/ulcerative colitis (UC)

Subjects with inflammatory bowel disease (IBD) may experience abdominal cramps and pain, diarrhea that may be bloody, urgency to defecate, constipation, nausea, vomiting, fever, weight loss, loss of appetite, and/or iron deficiency anemia. due to blood loss. IBD symptoms can occur in flares, with alternating periods of symptomatic and asymptomatic illness. IBD can be diagnosed by a combination of tests, including stool examinations (to eliminate the possibility of infectious causes of diarrhea, detect trace amounts of blood in stool, and quantify biomarkers associated with IBD, such as fecal calprotectin), a blood count comprehensive to assess levels of inflammation, blood tests to assess biomarkers including C-reactive protein (CRP) and perinuclear anti-neutrophil cytoplasmic antibody (pANCA), barium x-ray, sigmoidoscopy, colonoscopy and endoscopy. Patient populations include subjects with ulcerative colitis (UC; limited to the colon or large intestine), subjects with Crohn's disease (CD; affecting any segment of the gastrointestinal tract), and subjects with different disease severities (mild, moderate, serious).

Type 2 Diabetes/NASH/NAFLD

In some embodiments, subjects with type 2 diabetes may be treated according to the methods provided herein. Subjects with type 2 diabetes may experience blurred vision, peripheral neuropathy, increased urination, increased thirst, fatigue, increased hunger, weight loss, or yeast, bladder, kidney, skin, or other infections. Type 2 diabetes is diagnosed based on criteria outlined by the American Diabetes Association (ADA), including the following: fasting plasma glucose (FPG) of 126 mg/dL (7 mM) or higher, or a plasma glucose level at 2 hours of 200 mg/dL (11.1 mM) or more during a 75 g oral glucose tolerance test (OGTT), or a random plasma glucose of 200 mg/dL (11.1 mM) or more in a patient with classic symptoms of hyperglycemia or hyperglycemic crisis, or a hemoglobin A1c (HbA1c) level of 6.5% or more. Patient populations include adults and children with type 2 diabetes, subjects at risk of developing type 2 diabetes (e.g., subjects with prediabetes or overweight subjects), and subjects with type 2 diabetes along with metabolic syndrome conditions including obesity, stress high blood pressure, elevated serum triglycerides, and low high-density lipoprotein (HDL) levels.

In some embodiments, subjects presenting with nonalcoholic fatty liver disease (NAFLD) and/or nonalcoholic steatohepatitis (NASH) may be treated according to the methods provided herein.

Nonalcoholic fatty liver disease (NAFLD) is characterized by an abnormal accumulation of fat in the liver. NAFLD can progress to nonalcoholic steatohepatitis (NASH), which is characterized by liver inflammation, fibrosis, and cirrhosis. Subjects with NAFLD may be asymptomatic. Subjects with NAFLD or NASH may present with liver enlargement (seen during physical examination), fatigue, weight loss, general weakness, and/or pain in the right upper abdomen. The diagnosis of NAFLD/NASH includes elevated blood levels of alanine aminotransferase (ALT) or aspartate aminotransferase (AST), liver enlargement, and specific histopathologic markers (e.g., by liver biopsy, abdominal ultrasound, CT scan, or MRI). Patient populations include subjects with NAFLD, subjects with NASH, subjects at risk of developing NAFLD/NASH (e.g., subjects who are overweight or have high cholesterol levels), and subjects with NAFLD/NASH along with metabolic syndrome conditions including obesity, elevated fasting plasma glucose, elevated blood pressure, elevated serum triglycerides, and low high-density lipoprotein (HDL) levels. Obesity

In some embodiments, obese subjects may be treated according to the methods provided herein. Obesity is a major health problem and can have a negative effect on health. For example, obesity can reduce life expectancy and/or increase health problems, such as diabetes, high blood pressure, heart disease, stroke, high cholesterol, sleep apnea, and arthritis. Obese subjects have a body mass index (BMI) greater than 30 kg/m2. Alternatively, obese subjects can be classified based on body fat percentage (more than 25% for men or more than 33% for women). Diagnosis may also include an evaluation of fasting lipid levels (cholesterol, triglycerides), liver function, glucose levels, insulin levels, glycated hemoglobin (HbA1c), and/or glucose tolerance. Patient populations include subjects with childhood obesity, moderate obesity, morbid/severe obesity, genetic causes of obesity (including Prader-Willi syndrome, Bardet-Biedl syndrome, Cohen syndrome, and MOMO syndrome), and obesity along with other health conditions. metabolic syndrome (elevated blood pressure, elevated fasting plasma glucose, elevated serum triglycerides, and low high-density lipoprotein (HDL) levels).

Colitis induced by Clostridium difficile infection (CDI)

In some embodiments, subjects with Clostridium difficile infection (CDI)-induced colitis may be treated according to the methods provided herein. Subjects with CDI-induced colitis may present with watery diarrhea, cramps, abdominal pain, anorexia, malaise, fever, dehydration, lower abdominal tenderness, and/or rebound tenderness. The presence ofC. difficile in patients' stool can be tested by stool culture, glutamate dehydrogenase enzyme immunoassay, PCR assay to detect C. toxin genes. difficile, stool cytotoxin assay or enzyme immunoassay for C toxins A and B. difficile.Patient populations include subjects with primary CDI, subjects with recurrent CDI, subjects with different severity of CDI-associated diarrhea (mild, moderate, severe), and subjects at risk of CDI due to the presence of risk factors such as treatment with antibiotics, treatment with broad-spectrum antibiotics, residence in a hospital or long-term care facility, gastrointestinal tract surgery, colon diseases, weakened immune system, chemotherapy, advanced age, kidney disease, or use of pump inhibitors protons. Conventional care treatments for CDI include antibiotics such as metronidazole, fidaxomicin, or vancomycin. Treatments may also include probiotics, fecal transplant, and fluids to prevent dehydration. Resolution of the disease is measured by the decrease in diarrhea (for example, the absence of a 24-hour period with more than three unformed bowel movements) and the resolution of other symptoms described above. Clearance of infection can be verified by the absence of a positive stool test for C. difficile.

In one embodiment, methods are provided to prevent, treat, improve symptoms and/or prevent initial colonization or relapse of colonization by pathogens. In some embodiments, relapse occurs during or after the first-line treatment regimen or conventional care. In some cases, the pathogen load may initially decrease with conventional treatment, but then begins to increase again, potentially triggering a disease relapse. In some embodiments, glycan polymer preparations may be administered (e.g., at the beginning, during or after the initial treatment regimen) to prevent relapse or treat one or more symptoms of relapse. In some embodiments, the bacteria, pathobionts or disease-associated pathogens are selected from the group consisting of the species Bilophila wadsworthia, Campylobacter jejuni, Citrobacter farmer, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Collinsella aerofaciens, Enterobacter hormaechei, Enterococcus faecalis, Enterococcus faecium , Escherichia coli, Fusobacterium varium, Fusobacterium nucleatum, Haemophilus parainfluenzae, Klebsiella pneumonia, Peptostreptococcus stomatis, Porphyromonas asaccharolytica, Pseudomonas aeruginosa, Salmonella bongori, Salmonella enteric, Shigella boydii, Shigella Dysenteriae, Shigella flexneri, Shigella sonnei, aureus, Streptococcus infantarius, Vibrio cholera and Yersinia enterocolitica.

In some embodiments, the disease-associated bacteria, pathobionts or pathogens include the genera Bilophila, Campylobacter, Candidatus, Citrobacter, Clostridium, Collinsella, Desulfovibrio, Enterobacter, Enterococcus, Escherichia, Fusobacterium, Haemophilus, Klebsiella, Lachnospiraceae, Peptostreptococcus, Porphyromonas, Portiera, Providencia , Pseudomonas, Salmonella, Shigella, Staphylococcus, Streptococcus, Vibrioy Yersinia.

Colonization by vancomycin-resistant enterococci (VRE)

In some embodiments, subjects exhibiting colonization and infection with vancomycin-resistant enterococci (VRE) may be treated according to the methods provided herein. Bacteria of the genus Enterococcus are common members of the intestinal microbiota. Vancomycin-resistant members of this genus, commonly E. faecalisyE. faecium, can cause colonization and infection by vancomycin-resistant enterococci (VRE). Subjects colonized by VRE may have a stool sample, rectal swab, perirectal swab, or sample from another body site positive for VRE. Vancomycin resistance can be tested by bacterial culture or by PCR-based assays that detect operons of the vancomycin resistance gene (Van). Although colonized subjects may be asymptomatic, this population is at increased risk for VRE infection. Subjects with VRE infection may present with diarrhea, fever, chills, urinary tract infection (UTI), bacteremia, endocarditis, intra-abdominal and pelvic infection, respiratory infection, or infection elsewhere in the body. Patient populations include subjects who are colonized by VRE, subjects who have a VRE infection, and subjects who are at risk for VRE colonization or infection due to the presence of risk factors such as hospitalization, nursing home residence, long-term, long-term use of antibiotics, immunosuppression, surgery, open wounds, indwelling devices (for example, intravenous lines or urinary catheters), or employment as a healthcare worker.

Atopic dermatitis (AD)

In some embodiments, subjects with atopic dermatitis (AD) may be treated according to the methods provided herein. Subjects with atopic dermatitis (AD) may present with dry, itchy, and/or inflamed skin. The diagnosis and severity of AD can be determined by the SCORAD index (Oranje, AP,et al.British Journal of Dermatology 157.4 (2007): 645-648) or the Eczema Area and Severity Index (EASI) score (Hanifinet al .,Experimental Dermatology, 2001, 10:11). AD can occur in flares, with alternating periods of symptomatic and asymptomatic disease. Staphylococcus aureus is commonly present in AD skin sites, and biomarkers including IgE and inflammatory cytokines and chemokines or Th2 may also be elevated in diseased or affected skin. systemically. Patient populations include infants with early-onset AD, children with pediatric AD, adults with late-onset AD, pregnant women at risk for AD flares (“atopic eruption of pregnancy”), subjects with mild, moderate AD flares or severe, or subjects who are at risk of developing AD.

Asthma

In some embodiments, subjects with asthma may be treated according to the methods provided herein. Subjects with asthma may experience wheezing, cough, difficulty breathing, and/or chest tightness or pain. These symptoms are commonly episodic and can be triggered by factors such as exercise or exposure to allergens. Additionally, children with asthma may have a history of recurrent bronchitis, bronchiolitis, or pneumonia or persistent coughs with colds. The diagnosis of asthma is established by pulmonary function testing with spirometry in the presence and absence of treatment with a bronchodilator. Patient populations include infants with asthma; subjects with childhood asthma; adult-onset asthma; intermittent, mild persistent, moderate persistent, or severe persistent asthma; exercise-induced asthma; Allergic asthma; variant asthma with cough; occupational asthma; nocturnal asthma; and subjects who are at risk of developing asthma, for example, due to a family history of atopy.

inflammatory diseases

In some embodiments, administration of the glycan polymer preparation reduces inflammation. In some embodiments, a subject is identified as suitable for treatment if the subject has or is suspected of having a disease, disorder or condition including: inflammatory gastrointestinal diseases including inflammatory bowel disease (IBD), ulcerative colitis (UC) , Crohn's disease (CD), idiopathic inflammation of the small intestine, indeterminate colitis, pouchitis; irritable bowel syndrome (IBS), colon and liver cancers, necrotizing enterocolitis (NEC), intestinal inflammation, constipation, microscopic colitis, diarrhea; graft versus host disease (GVHD); allergies (food); pseudomembranous colitis; indigestion or non-ulcer dyspepsia; diverticulosis or diverticulitis, ischemic colitis; radiation colitis or enteritis; collagenous colitis; stomach flu; and polyps.

In one embodiment, the subject who is identified as suitable for treatment with a glycan polymer preparation has or is suspected of having inflammatory bowel disease (IBD), ulcerative colitis (UC), Crohn's disease (CD), intestinal inflammation , microscopic colitis or similar disease, disorder or condition that is associated with inflammation of the intestine.

In one embodiment, the subject who is identified as suitable for treatment with a glycan polymer preparation has or is suspected of having idiopathic inflammation of the small intestine, indeterminate colitis, pouchitis, pseudomembranous colitis, ischemic colitis, radiation colitis (enteritis). , collagenous colitis or similar disease, disorder or condition that is associated with inflammation of the intestine.

In one embodiment, the subject who is identified as suitable for treatment with a glycan polymer preparation has or is suspected of having gastroenteritis; graft versus host disease (GVHD) or an allergy (food).

In one embodiment, the subject who is identified as suitable for treatment with a glycan polymer preparation has or is suspected of having irritable bowel syndrome (IBS), constipation, diarrhea, indigestion, non-ulcer dyspepsia or disease, disorder or condition. similar that is associated with altered intestinal transit.

In one embodiment, the subject who is identified as suitable for treatment with a glycan polymer preparation has or is suspected of having colon cancer, liver cancers, necrotizing enterocolitis (NEC); diverticulosis or diverticulitis; polyps or similar disease, disorder or condition that is associated with structural alteration of the intestine.

Metabolic diseases

In some embodiments, a subject is identified as suitable for treatment if the subject has or is suspected of having a disease, disorder or condition including: obesity, prediabetes, type II diabetes, high blood cholesterol, high LDL, blood pressure high, high fasting blood sugar, high triglycerides, low HDL nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH); metabolic syndrome; hyperammonemia, deficiency of essential nutrients, hemochromatosis, lactose intolerance, gluten intolerance; and acrodermatitis enteropathica.

In one embodiment, the subject who is identified as suitable for treatment with a glycan polymer preparation has or is suspected of having obesity, prediabetes (insulin resistance), type I diabetes, high blood sugar levels. fasting (hyperglycemia), metabolic syndrome or similar disease, disorder or condition that is associated with symptoms of metabolic diseases.

In one embodiment, the subject who is identified as suitable for treatment with a glycan polymer preparation has or is suspected of having high blood cholesterol, high LDL, high blood pressure (hypertension), high triglyceride levels, low HDL or a similar cardiovascular risk factor.

In one embodiment, the subject who is identified as suitable for treatment with a glycan polymer preparation has or is suspected of having nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), hyperammonemia or disease, disorder or similar liver condition. In one embodiment, the subject who is identified as suitable for treatment with a glycan polymer preparation has or is suspected of having lactose intolerance, gluten intolerance, or a similar disease, disorder or condition that is associated with food intolerance.

In one embodiment, the subject who is identified as suitable for treatment with a glycan polymer preparation has or is suspected of having essential nutrient deficiency, hemochromatosis, acrodermatitis enteropathica, or similar disease, disorder or condition that is associated with poor management. of nutrients.

In one embodiment, a method of treating a metabolic disorder in a human being in need thereof is provided by: administering to the human a glycan polymer preparation composition for treating the metabolic disorder. In one embodiment, the metabolic disorder is selected from obesity, adiposity, insulin resistance, diabetes and fatty liver syndrome.

Metabolic disorders may include disorders, diseases and conditions caused or characterized by abnormal weight gain; energy use or consumption; altered responses to nutrients, energy sources, hormones or other signaling molecules; or altered metabolism of carbohydrates, lipids, proteins or nucleic acids, or a combination thereof. Examples of metabolic disorders include insulin resistance, insulin sensitivity, fatty liver syndrome, obesity, adiposity, and diabetes (e.g., type 1 diabetes, type 2 diabetes). In a variation, the methods provided herein treat obesity. Provided herein are methods of treating obesity in a subject in need thereof using a glycan polymer preparation composition that can alter the intestinal microbiota of the subject in a manner that results in weight loss and/or decreased body fat in the subject.

In one embodiment, a method of reducing adiposity in a subject in need is provided by: administering to the human a glycan polymer preparation composition in an amount effective to reduce adiposity. Adiposity may be determined using any appropriate method known in the art, including, for example, waist circumference, waist-hip ratio, skinfold thickness, bioelectrical impedance, underwater weighing, air displacement plethysmography, or hydrometry. In one embodiment, a method of improving glucose metabolism in a subject in need thereof is provided by: administering to the subject a glycan polymer preparation composition in an amount effective to improve glucose metabolism. Glucose metabolism can be determined by any appropriate method known in the art, including, for example, fasting blood sugar level, fasting insulin level, postprandial blood sugar test, postprandial insulin test, tolerance test. to oral glucose, intravenous glucose tolerance test, glycated hemoglobin level or random blood sugar test.

In one embodiment, a method of increasing insulin sensitivity in a human is provided by: administering to the subject a glycan polymer preparation composition in an amount effective to increase insulin sensitivity, wherein the human has an insulin sensitivity before administration of the glycan polymer preparation and an insulin sensitivity after administration of the glycan polymer preparation, and the insulin sensitivity of the human after administration of the glycan polymer preparation is greater than the insulin sensitivity of the human before administration of the glycan polymer preparation. Insulin sensitivity may be determined by any appropriate method known in the art, including, for example, fasting blood sugar level, fasting insulin level, postprandial blood sugar test, postprandial insulin test, tolerance test. to oral glucose, intravenous glucose tolerance test, glycated hemoglobin level or random blood sugar test.

Infectious diseases

In some embodiments, administration of the glycan polymer preparation reduces infection. In some embodiments, a subject is identified as suitable for treatment if the subject has or is suspected of having a disease, disorder or condition including: gastrointestinal infectious diseases including Clostridium difficile infection (CDI); vancomycin-resistant enterococci (VRE) infection, infectious colitis, and C. colitis. difficile; mycosis, such as, for example, Candida albicans infection, Campylobacter jejuni infection, Helicobacter pylori infection; diarrhea, such as, for example, Clostridium difficile-associated diarrhea (CDAD), antibiotic-associated diarrhea (AAD), antibiotic-induced diarrhea, diarrhea traveler's disease (TD), pediatric diarrhea, infectious (acute) diarrhea, colon and liver cancer, amoeboma; necrotizing enterocolitis (NEC) and small intestinal bacterial overgrowth (SIBO); indigestion or non-ulcer dyspepsia; anal fissures, perianal abscess and anal fistula; diverticulosis or diverticulitis; peptic ulcers; and gastroenteritis.

In one embodiment, the subject who is identified as suitable for treatment with a glycan polymer preparation has or is suspected of having a Clostridium difficile infection (CDI); a vancomycin-resistant enterococci (VRE) infection, infectious colitis, or C. colitis. difficile.

In one embodiment, the subject who is identified as suitable for treatment with a glycan polymer preparation has or is suspected of having mycoses, such as, for example, Candida albicans infection, Campylobacter infection, or Helicobacter pylori infection.

In some embodiments, the GI tract infection is a bacterial or viral infection, such as an infection with, for example, VRE,C. difficile, Escherichia coli, Salmonella, Shigella, Campylobacter, Vibrio cholera, Clostridium perfringes, Bacillus cereus, Vibrio parahemolyticus, Yersinia enterocolitica, Helicobacter pylori, rotavirus or norovirus.

In some embodiments, the GI tract infection is a fungal infection, such as an infection with, for example, Candida, Aspergillus, Mucor, Cryptococcus, Histoplasma or Coccidioides.

In some embodiments, the gastrointestinal tract infection is a protozoan infection, such as an infection with, for example, Entamoeba histolytica, Giardia lamblia, Cryptosporidium parvum.

In one embodiment, the subject who is identified as suitable for treatment with a glycan polymer preparation has or is suspected of having diarrhea, such as, for example, Clostridium difficile associated diarrhea (CDAD), antibiotic associated diarrhea (AAD). , antibiotic-induced diarrhea, traveler's diarrhea (TD), pediatric diarrhea, or infectious (acute) diarrhea.

In one embodiment, the subject who is identified as suitable for treatment with a glycan polymer preparation has or is suspected of having necrotizing enterocolitis (NEC); stomach flu; small intestine bacterial overgrowth (SIBO) or similar disease, disorder or condition that is associated with an infection of the GI tract.

In one embodiment, the subject who is identified as suitable for treatment with a glycan polymer preparation has or is suspected of having colon cancer, liver cancer, amoeboma; indigestion or non-ulcer dyspepsia; anal fissures, perianal abscess and anal fistula; diverticulosis or diverticulitis; peptic ulcer or similar disease, disorder or condition that is associated with structural alterations of the GI tract.

Other diseases

In some embodiments, a subject is identified as suitable for treatment if the subject has or is suspected of having a disease, disorder or condition including: autoimmune arthritis, type I diabetes, atopic dermatitis, autism, asthma, cardiovascular disease, chronic kidney disease, multiple sclerosis, karyopathies, psoriasis, hyperammonemia, hepatic encephalopathy, cachexia, gout, drug intolerance (for example, metformin), low oral bioavailability of drugs, fecal incontinence, Hirschsprung's disease, anismus, colic, ileus, hemorrhoids and intestinal intussusception.

In one embodiment, the subject who is identified as suitable for treatment with a glycan polymer preparation has or is suspected of having autoimmune arthritis, type I diabetes, multiple sclerosis, psoriasis or similar autoimmune disease, disorder or condition.

In one embodiment, the subject is identified as suitable for treatment with a glycan polymer preparation or is suspected of having asthma, atopic dermatitis, or similar environmentally driven allergy.

In one embodiment, the subject who is identified as suitable for treatment with a glycan polymer preparation has or is suspected of having chronic kidney disease, heart disease, cardiovascular disease or similar disease, disorder or condition associated with organ failure.

In one embodiment, the subject who is identified as suitable for treatment with a glycan polymer preparation has or is suspected of having autism, hyperammonemia, hepatic encephalopathy, or similar disease, disorder or condition that is associated with neurological symptoms.

In one embodiment, the subject who is identified as suitable for treatment with a glycan polymer preparation has or is suspected of having cachexia, gout, or a similar nutritional disorder.

In one embodiment, the subject who is identified as suitable for treatment with a glycan polymer preparation has or is suspected of having Hirschsprung's disease, ileus, anismus, intussusception, fecal incontinence, hemorrhoids, or similar gastrointestinal disorder.

Treatment effects

In some embodiments, the subject experiences a reduction in at least one symptom of a disease or disorder following treatment. In some embodiments, a reduction in the severity of a symptom after treatment can be determined (e.g., by measuring a known biomarker) and is on the order of about 3%, 5%, 7%, 10%, 20%, 30%. , 40%, 50%, 60%, 70%, 80%, 90%, 95% or approximately 100%. In some embodiments, the symptoms, measured as described herein, decrease by an average of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%. , 95% or approximately 100% compared to symptoms before administration of a glycan polymer preparation. In some embodiments, the reduction in symptom severity persists for at least about one day, two days, three days, four days, five days, one week, two weeks, three weeks, one month, 3 months, 6 months, 9 months, one year, two years, five years, ten years after treatment or the reduction is permanent.

In one embodiment, the severity of a symptom of a disease, disorder or condition described herein remains partially, substantially or completely eliminated or diminished in a subject for at least about 1 day, 1 week, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, one year, 18 months, two years, three years, four years, five years, ten years or more than ten years after the end of treatment. In another embodiment, a symptom of a disease, the severity of a disorder or condition described herein is permanently eliminated or decreased in a subject after completion of treatment.

In some embodiments, administration of the glycan polymer preparations improves the overall health of the host and/or the health of a specific niche, such as the GI tract, for example, by modulating (e.g., increasing or decreasing) the growth or the abundance of one or more members of the microbial community in the niche (such as resident commensal bacteria and/or acquired pathogens or pathobionts). Glycan polymer preparations, when administered to a subject in an effective amount, can modulate one or more host pathways. Treatment with the glycan polymer preparation may result in increases or decreases in one or more biomarkers that can be determined by methods known in the art. A researcher can easily determine at what point(s) during treatment the biomarker(s) should be measured, for example, before treatment, at various intervals during treatment, and/or after treatment. Any suitable sample, for example a specific gastrointestinal sample such as, for example, a tissue sample or biopsy, a swab, a gastrointestinal secretion (such as feces/a stool sample), etc., may be collected from the subject. and the sample can be analyzed. In some embodiments, a substantial increase or decrease in a biomarker may be detected. In some embodiments, the glycan polymer preparation is digested by intestinal microbiota (e.g., clostridia), resulting in, for example, the release of short chain fatty acids such as butyrate, acetate and propionate, which can act with an immunomodulatory capacity (for example, anti-inflammatory) and other metabolites (for example, bile acids and lactate) that can confer beneficial effects on the health of the host.

To evaluate the effect of the administered glycan polymer preparation compositions on SCFA production in the intestine, fecal samples can be collected. Levels of SCFAs, particularly acetate, propionate, and butyrate, can be quantified. SCFAs, creatines, and hydroxy-SCFAs can be quantified by alkalizing stool samples, fingerprinting the metabolic composition of the sample using, for example, a 1H 1D NMR spectrometer, and analyzing with supervised multivariate statistical methods. Inulin can serve as a positive control.

In some embodiments, microbial metabolite profiles from patient samples or microbial cultures from subject samples are used to identify risk factors for developing a gastrointestinal infectious and/or inflammatory disease, disorder or condition. Exemplary metabolites for purposes of diagnosis, prognostic risk assessment, or treatment evaluation include those listed in Table 5. In some embodiments, microbial metabolite profiles are taken at different time points during the disease and treatment of a subject in order to better evaluate the pathological state of the subject including recovery or relapse events. Such monitoring is also important to reduce the risk of a subject developing a new gastrointestinal disease, disorder or condition. In some embodiments, metabolite profiles inform subsequent treatment.

Additionally, if determined to be useful by an attending physician or other healthcare provider, the glycan polymer preparation compositions described herein may be administered in combination with various other conventional healthcare therapies. In some embodiments, the combination of administration of the glycan polymer preparation and the conventional therapy agent has additive or synergistic treatment effects. Glycan polymer preparations may be administered before, simultaneously with, or after treatment with conventional care therapies. In some cases, therapies alter the composition and health of the normal microbiota of the GI tract (e.g., use of antibacterial, antiviral, or antifungal agents), which can lead to unwanted proliferation of harmful bacteria or pathogens, which may cause one or more of the symptoms described herein. In some embodiments, administration of the glycan polymer preparations described herein is useful in alleviating such symptoms and improving the composition of the gastrointestinal microbial community.

Combinations

Additional substances may be administered together with a glycan polymer preparation. In some embodiments, the glycan polymer preparation may also be combined with another agent (e.g., a therapeutic agent, micronutrient, prebiotic, probiotic, or synbiotic).

These substances can enhance the action of glycan polymer doses, for example, by promoting the growth of bacteria, for example, in the intestine that alleviate the symptoms of a disease, disorder (for example, described herein), increasing the adhesion of probiotic or beneficial commensal bacteria in the niche or in the intestine. These substances may be administered before treatment with a glycan polymer preparation, during treatment with a glycan polymer preparation, after treatment with a glycan polymer preparation, or any combination thereof. If administered during treatment with the glycan polymer preparation, they may be administered with the dose of glycan polymer preparation being administered, or before or after the dose of glycan polymer preparation, or any combination thereof. In one embodiment, substances for use together with a glycan polymer preparation include probiotic microbe(s), prebiotics, therapeutic agents or buffers/carriers/excipients. One or more of these substances may be used in combination with the glycan polymer preparation at any suitable time before, during, after treatment or some combination thereof.

In some embodiments, the additional agent is a therapeutic agent, for example, a drug that causes dysbiosis, for example, a drug that alters the normal growth of the gastrointestinal microbiota, for example, a chemotherapy drug, an antidiabetic drug, an immunosuppressive drug , an antimicrobial drug, an antipsychotic drug, a proton pump inhibitor drug, or a nonsteroidal anti-inflammatory drug (NSAID). The glycan polymer preparation, in some embodiments, reduces symptoms induced by the drug or treatment in a human subject. Symptoms include digestive abnormalities, such as, for example, weight gain, constipation, heartburn, upset stomach, gas, bloating, flatulence, diarrhea, abdominal pain, cramps, nausea and vomiting.

In some embodiments, the additional agent is a micronutrient. In some embodiments, the micronutrient is selected from the group consisting of a trace element, choline, a vitamin, and a polyphenol. In some embodiments, the micronutrient is a trace element. Trace elements suitable as micronutrients include, but are not limited to, boron, cobalt, chromium, calcium, copper, fluorine, iodine, iron, magnesium, manganese, molybdenum, selenium and zinc. In some embodiments, the micronutrient is a vitamin. Vitamins suitable as micronutrients include, but are not limited to, vitamin B complex, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxal, pyridoxamine), vitamin B7 (biotin), vitamin B8 (ergadenylic acid), vitamin B9 (folic acid), vitamin B12 (cyanocobalamin), choline, vitamin A (retinol), vitamin C (ascorbic acid), vitamin D, vitamin E ( tocopherol), vitamin K, carotenoids (alpha carotene, beta carotene, cryptoxanthin, lutein, lycopene) and zeaxanthin.

In some embodiments, the micronutrient is a polyphenol. Polyphenols are chemical compounds or molecules that are characterized by having at least one aromatic ring with one or more hydroxyl groups. In some embodiments, the polyphenol is a synthetic polyphenol or a naturally occurring polyphenol. In some embodiments, the polyphenol is a naturally occurring polyphenol derived from plant material. In some embodiments, the polyphenol is a flavonoid or a catechin. In some embodiments, the flavonoid or catechin is selected from anthocyanins, chalcones, dihydrochalcones, dihydroflavonols, flavanols, flavanones, flavones, flavonols and isoflavonoids. In some embodiments, the polyphenol is a lignan. In some embodiments, the polyphenol is selected from alkylmethoxyphenols, alkylphenols, curcuminoids, furanocoumarins, hydroxybenzaldehydes, hydroxybenzoketones, hydroxycinnamaldehydes, hydroxycoumarins, hydroxyphenylpropenes, methoxyphenols, naphthoquinones, phenolic terpenes and tyrosols. In some embodiments, the polyphenol is a tannin or tannic acid. In some embodiments, the polyphenol is selected from hydroxybenzoic acids, hydroxycinnamic acids, hydroxyphenylacetic acids, hydroxyphenylpropanoic acids, and hydroxyphenylpentanoic acids. In some embodiments, the polyphenol is a stilbene.

In some embodiments, the pharmaceutical compositions and medical foods and dietary supplements comprising glycan polymer preparations described herein further comprise a prebiotic substance or a preparation thereof.

In some embodiments, prebiotics may be administered to a subject receiving the pharmaceutical compositions or medical foods or dietary supplements comprising glycan polymer preparations described herein. Prebiotics are nondigestible substances that, when consumed, can provide a beneficial physiological effect on the host by selectively stimulating the growth or favorable activity of a limited number of indigenous bacteria in the intestine (Gibson G R, Roberfroid M B. J Nutr . June 1995; 125(6): 1401-12. A prebiotic such as a dietary fiber or a prebiotic oligosaccharide (e.g., crystalline cellulose, wheat bran, oat bran, corn fiber, soybean fiber, beet fiber and the like) may further stimulate the growth of probiotic bacteria and/or or commensals in the intestine by providing a fermentable dose of carbohydrates to bacteria and increasing the levels of those microbial populations (e.g., lactobacilli and bifidobacteria) in the gastrointestinal tract.

Prebiotics include, but are not limited to, various galactans and carbohydrate-based gums, such as psyllium, guar, carrageenan, gellan, lactulose and konjac. In some embodiments, the prebiotic is one or more of galactooligosaccharides (GOS), lactulose, raffinose, stachyose, lactosucrose, fructooligosaccharides (FOS, for example, oligofructose or oligofructan), inulin, isomaltooligosaccharide, xylo-oligosaccharides (XOS), paratinose oligosaccharide , isomaltose oligosaccharides (IMOS), transgalactosylated oligosaccharides (e.g., transgalactooligosaccharides), transgalactosylated disaccharides, soybean oligosaccharides (e.g., soybean oligosaccharides), chitosan oligosaccharides (chioses), gentiooligosaccharides, soybean and pectin oligosaccharides, glucooligosaccharides, pecticooligosaccharides, polycondensates palatinose, fructose anhydride III, sorbitol, maltitol, lactitol, polyols, polydextrose, linear and branched dextrans, pulalan, hemicelluloses, reduced paratinose, cellulose, betaglucose, betagalactose, betafructose, verbascose, galactinol, xylan, inulin, chitosan, betaglucan, guar gum, gum arabic, pectin, high sodium alginate and lambda carrageenan, or mixtures thereof.

Prebiotics can be found in certain foods, for example, chicory root, Jerusalem artichoke, dandelion leaves, garlic, leek, onion, asparagus, wheat bran, wheat flour, banana, milk, yogurt, sorghum, burdock, broccoli, brussels sprouts, cabbage, cauliflower, collard greens, kale, radish and kohlrabi and miso. In some embodiments, the glycan polymers described herein are administered to a subject along with a diet that includes prebiotic-rich foods. Suitable sources of soluble and insoluble fibers are commercially available.

In some embodiments, the pharmaceutical compositions and medical foods and dietary supplements comprising glycan polymer preparations further comprise a probiotic bacteria or preparation thereof, e.g., derived from bacterial cultures generally recognized as safe (GRAS) or commensal microbes. or known probiotics. In some embodiments, to maximize the beneficial effect of endogenous commensal microbes or exogenously administered probiotic microorganisms, pharmaceutical compositions and medical foods and dietary supplements comprising glycan polymer preparations are administered to stimulate growth and/or activity. of advantageous bacteria in the GI tract.

Examples of suitable probiotics include, but are not limited to, organisms classified as genera Bacteroides, Blautia, Clostridium, Fusobacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, Akkermansia, Faecalibacterium, Roseburia, Prevotella, Bifidobacterium, Lactobacillus, Bacillus, Enterococcus, Escherichia, Streptococcus, Saccharomyces, Streptomyces, and Christensenellaceae family. Non-exclusive examples of probiotic bacteria that can be used in the methods and compositions described herein include L. acidophilus, Lactobacillus species, such as L. crispatus, L. casei, L. rhamnosus, L. reuteri, L. fermentum, L. plantarum, L. sporogenesyL. bulgaricus, as well as Bifidobacterum species, such as B. lactis, B. animalis, B. bifidum, B. longum, B. adolescentis, and B. infantis.Yeasts such as Saccharomyces boulardii are also suitable as probiotics for intestinal administration, for example by means of oral dosage forms or foods. For example, yogurt is a product that already contains species of bacteria, such as Lactobacillus bulgaricus and Streptococcus thermophilus.

Beneficial bacteria for modulating the gastrointestinal microbiota may include bacteria that produce organic acids (lactic and acetic acids) or that produce cytotoxic or cytostatic agents (to inhibit pathogen growth), such as, for example, hydrogen peroxide (H2O2). and bacteriocins. Bacteriocins are small antimicrobial peptides that can kill closely related bacteria or exhibit a broader spectrum of activity (e.g., nisin).

Beneficial bacteria may include one or more of the genus Akkermansia, Anaerophilum, Bacteroides, Blautia, Bifidobacterium, Butyrivibrio, Clostridium, Coprococcus, Dialister, Dorea, Fusobacterium, Eubacterium, Faecalibacterium, Lachnospira, Lactobacillus, Phascolarctobacterium, Peptococcus, Peptostreptococcus, Prevotella, Roseburia, ptococcus ,and/or one or more of the species Akkermansia municiphilia, minuta, Clostridium coccoides, Clostridium leptum, Clostridium scindens, Dialister invisus, Eubacterium rectal, Eubacterium eligens, Faecalibacterium prausnitzii, Streptococcus salivarius and Streptococcus thermophilus.

In some embodiments, combinations are provided comprising a bacterial taxon selected from column 1 of Tables 19, 20, or 21 and a glycan preparation described herein. In some embodiments, the combination preparation comprises a microbial preparation of a microbe selected from column 1 of Tables 19, 20 or 21 and a glycan preparation selected from columns 3-10 (Table 19) or columns 2-9 ( tables 20 and 21). In some embodiments, synbiotic combinations are provided suitable for administration to a human subject in need (e.g., oral or rectal administration). In some embodiments, the bacterial taxa selected for the combination are spore-forming bacterial taxa. In some embodiments, the glycan preparation selected for combination is a (fermentable) substrate (e.g., for a glycosidase enzyme) of spore-forming bacterial taxa.

Furthermore, if desired, pharmaceutical compositions and medical foods and dietary supplements comprising glycan polymer preparations may comprise therapeutically active agents, prebiotic substances and/or probiotic bacteria. Alternatively or in addition, the therapeutically active agents, prebiotic substances and/or probiotic bacteria may be administered separately (e.g., before, simultaneously with or after the administration of the glycan polymers) and not as part of the pharmaceutical composition or medical food. or dietary supplement (e.g., as a coformulation) of glycan polymers. In some embodiments, the pharmaceutical compositions or medical foods or dietary supplements comprising glycan polymer preparations are administered in combination with a recommended or prescribed diet, for example, a diet rich in foods containing probiotics and/or prebiotics, such as determined by a doctor or other health professional. Therapeutically active agents, prebiotic substances and/or probiotic bacteria may be administered to modulate the subject's gut microbiome. In some embodiments, the combined effect (e.g., on the number or intensity of microbial, genomic or functional changes) is additive. In other embodiments, the combined effect (for example, on the number or intensity of microbial, genomic or functional changes) is synergistic.

Administration of glycan polymer preparations

For any glycan polymer preparation composition used in a method described herein (for example, a method of treating a disease, disorder or condition listed in Table 5), a therapeutically effective dose can be initially estimated from laboratory animal models known to those skilled in the art. Such information can be used to more accurately determine useful doses in humans. Initial dosages can also be estimated from in vitro or in vivo data. Initial dosages can also be formulated by comparing the efficacy of compounds used in the methods described herein in model trials with the efficacy of known compounds. For example, initial dosages can be formulated by comparing the efficacy of glycan polymer preparations in model trials with the efficacy of other compounds that have demonstrated efficacy in the treatment of the present conditions. In this method, an initial dosage can be obtained by multiplying the ratio of effective concentrations obtained in the model test for the glycan polymer preparations used in the methods described herein and the control compound by the effective dose of the control compound. For example, if a preparation useful in the present method is twice as effective in a model test as a known compound (for example, the effective concentration (EC50) of the glycan polymer preparation is equal to half the EC50 of the known compound in the same assay), an initial effective dosage of the glycan polymer preparation would be half of the known dosage for the known compound. Using these initial guidelines, one of ordinary skill in the art can determine an effective dosage in subjects, such as humans. The dosage amount and interval can be individually adjusted to provide levels of the glycan polymer preparation that are sufficient to maintain the therapeutic effect. One skilled in the art will be able to optimize therapeutically effective local dosages without undue experimentation.

Depending on the disorder and the subject to be treated and the route of administration, the compositions can be administered in variable doses. In one embodiment, the smallest effective amount or dose of glycan polymer preparation is used. In some embodiments, the glycan polymer preparation is administered at a dose of from about 0.01 mg/kg to about 10,000 mg/kg, from about 0.1 mg/kg to about 1,000 mg/kg, from about 1 mg /kg to about 100 mg/kg, from 0.05 mg/kg to about 5,000 mg/kg, from about 0.5 mg/kg to about 5,000 mg/kg, from about 5 mg/kg to about 500 mg/kg . This dose may be given as mg/kg/day and may be given as a starting dose or may be increased or decreased over time (e.g., days or weeks) to reach a final dose.

In some embodiments, the glycan polymer preparation is administered at a total daily dose per subject of from about 1 mg per day to about 100 grams per day; from about 10 mg per day to about 10 grams per day; from approximately 100 mg per day to approximately 10 grams per day; from about 1 gram per day to about 10 grams per day, from about 2 grams per day to about 20 grams per day; from about 5 grams per day to about 50 grams per day, from about 10 grams per day to about 100 grams per day, from about 10 grams per day to about 50 grams per day, from about 10 grams per day to about 75 grams per day day, from approximately 20 grams per day to approximately 100 grams per day, from approximately 20 grams per day to approximately 50 grams per day, from approximately 20 grams per day to approximately 75 grams per day, from approximately 20 grams per day to approximately 100 grams per day, from approximately 50 grams per day to approximately 150 grams per day or from approximately 50 grams per day to approximately 200 grams per day.

In some embodiments, a symptom of a gastrointestinal disease, disorder or condition in a subject exhibiting the symptoms is decreased or eliminated by administering increasing, decreasing or constant amounts (or doses) of a glycan polymer preparation composition over a period of time to the subject. time frame. (for example, a treatment period).

In one embodiment, the composition contains beneficial, commensal and/or probiotic bacterial strains in an amount between 1 x 107 and 1*1013 CFU/dose and bacterial strain, or between 1*109 and 1x1011 CFU/dose and bacterial strain.

In some embodiments, the pharmaceutical composition is administered one, two or three times a day. In some embodiments, the pharmaceutical composition is administered twice daily. In some embodiments, the pharmaceutical composition is administered each day for a predetermined number of days (the treatment period). In some embodiments, the treatment period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 35, 42, 49, 56, 63, 70, 100, 200, 300 or 365 days. In some embodiments, the treatment period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. In some embodiments, the treatment period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 years or for life.

In one embodiment, the total duration of treatment periods for a gastrointestinal disease, disorder or condition may be approximately one day to 10 years, one day to 1 year, 1 day to 6 months, 1 day to 3 months. , from 1 day to 1 month, from one day to one week, from one day to five days, from one day to 10 days, from one week to approximately 12 weeks, or from approximately four weeks to approximately ten weeks, or from approximately four weeks to approximately eight weeks, or approximately six weeks. The subject may undergo an appropriate number of treatment periods, such as, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 treatment periods. During a treatment period, the subject takes a glycan polymer preparation composition described herein, optionally in conjunction with the ingestion of food products containing prebiotics and/or probiotics. In one embodiment, a glycan polymer preparation composition may also be administered in combination with another substance (such as a probiotic or a commensal beneficial bacteria, a prebiotic substance or a therapeutic agent), as described herein.

In some embodiments, the glycan polymer preparation composition may also be combined with an antibiotic that alters the normal growth of the gastrointestinal microbiota. Typically, the duration of antibiotic treatments is 1-14 days, 2-10 days or 5-7 days. In some embodiments, a glycan polymer preparation is administered to a subject in need thereof immediately after one or more antibiotic treatments have ended (e.g., 1 hour, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours). hours, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, or 4 weeks after antibiotic treatment has ended). During a course of antibiotic treatment, the glycan polymer preparation composition may be provided at the beginning of the antibiotic treatment; shortly after antibiotic treatment, for example, 1, 2, 3, 4, 5, 6, 7 or more days after treatment; or may be administered upon diagnosis of unwanted pathogen growth.

In some embodiments, the glycan polymer preparation composition may also be combined with a drug that causes dysbiosis, for example, a drug that alters the normal growth of the gastrointestinal microbiota, for example, a chemotherapy drug, an antidiabetic drug, a drug immunosuppressant, an antimicrobial drug, an antipsychotic drug, a proton pump inhibitor drug, or a nonsteroidal anti-inflammatory drug (NSAID). The glycan polymer preparation composition, in some embodiments, reduces symptoms induced by the drug or treatment in a human subject. Symptoms include digestive abnormalities, such as, for example, weight gain, constipation, heartburn, upset stomach, gas, bloating, flatulence, diarrhea, abdominal pain, cramps, nausea and vomiting. In some embodiments, a glycan polymer preparation is administered to a subject in need thereof immediately after one or more drug treatments have ended (e.g., 1 hour, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours , 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, or 4 weeks after antibiotic treatment has ended). During a course of drug treatment, the glycan polymer preparation composition may be provided prior to the start of drug treatment (e.g., 1, 2, 3, 4, 5, 6, 7 days before); the day of initiation of drug treatment; or shortly after antibiotic treatment, e.g., 1, 2, 3, 4, 5, 6, 7 or more days after treatment, and may optionally be provided only initially (e.g., for a short period) or throughout the entire course of treatment. duration of drug treatment, and may even be continued for a desired period after the drug treatment period has ended (e.g., for 1-7 days, 1-14 days, or 1-21 days later). In some embodiments, administration of the glycan polymer preparation composition is initiated or continued when one or more adverse effects (e.g., digestive abnormalities or pathogen growth) occur and/or are diagnosed in conjunction with drug treatment. In some embodiments, the treatment agent that causes a dysbiosis is not a drug but a radiation treatment or surgery and the glycan polymer preparation composition may also be administered as described herein.

In some embodiments, the total number and duration of treatment periods are based on a subject's response to treatment. For example, an individual may experience a reduction in symptoms after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days of treatment with a preparation composition of glycan polymer. In another example, an individual may experience a reduction in symptoms after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months of treatment with a glycan polymer preparation composition. . Therefore, the duration of treatment is determined by the response of an individual subject to a glycan polymer preparation composition and the occurrence of relief of one or more symptoms. Therefore, a subject may experience symptoms at a given dose of a glycan polymer preparation composition and may require the subject to remain at that dose, or at a lower dose, until the symptoms disappear. Therefore, in one embodiment, the duration of treatment is not determined from the beginning, but continues until the maximum dose of a glycan polymer preparation composition per day is reached, or until the desired level of reduction is achieved. of the symptoms. In one embodiment, the treatment is continuous.

In one embodiment, a subject may be administered a dose for the first treatment period during a treatment regimen and a second dose during a second treatment period. For example, a subject may be administered one dose of glycan polymer preparation composition over a period of one week and a second dose over a subsequent period of one week.

A subject may self-administer a glycan polymer preparation composition and the glycan polymer preparation composition is supplied or recommended (or prescribed) by a health care professional, for example, a physician or other qualified health care professional and, optionally , a healthcare professional monitors test results (for example, obtained for biomarkers from samples taken from the subject) and/or health changes and treatment endpoints. In some embodiments, the glycan polymer preparation composition is administered by a healthcare professional.

In one embodiment, a subject in need may undergo repeated courses of treatment with a glycan polymer preparation composition. The course of treatment may be repeated when symptoms reappear or increase to an unwanted level. Alternatively, the course of treatment may be repeated at regular or predetermined intervals. Thus, the treatment can be repeated after approximately one month, two months, three months, four months, six months, eight months, ten months, one year, 18 months, two years, three years, four years, five years or more. five years, or any combination thereof (for example, treatment may be repeated after one year and then every two to five years thereafter). Treatment may be repeated in the same manner (e.g., duration, dosage, timing of dosing, additional substances, etc.) as used in the first treatment or may be modified. For example, the duration of treatment can be shortened or lengthened, the dosage can be increased or decreased. Optionally, the treatment with the glycan polymer preparation may occur in combination with a number or different compositions of agents, for example, containing more or less other substances, or less or more substances (such as, for example, a prebiotic substance , a probiotic bacteria or a therapeutic agent) in addition to the glycan polymer preparation.

Additional substances may be administered together with a glycan polymer preparation composition. These substances may enhance the action of glycan polymer preparation doses, for example, by promoting the growth of bacteria in the GI tract that relieve symptoms of the gastrointestinal disease, disorder or condition, by increasing the adhesion of probiotic or beneficial commensal bacteria in the niche or in the intestine. These substances may be administered before treatment with a glycan polymer preparation, during treatment with a glycan polymer preparation, after treatment with a glycan polymer preparation, or any combination thereof. If administered during treatment with the glycan polymer preparation, they may be administered with the dose of glycan polymer preparation being administered, or before or after the dose of glycan polymer preparation, or any combination thereof. In one embodiment, substances for use together with a glycan polymer preparation composition include probiotic microbe(s), prebiotics, therapeutic agents or buffers/carriers/excipients. One or more of these substances may be used in combination with a glycan polymer preparation composition at any suitable time before, during, after treatment or some combination thereof.

Table 1: Characteristics of exemplary glycan polymers.

Table 2: Genus-level microbial constituents of the GI tract.

Table 3: Phyla and strains associated with metabolites as an example

Table 4: Prediction of CAZy glycoside hydrolase (GH) and glycosyltransferase (GT) family activity.

__________________________________

Table 5: Metabolites and associated indications

Table 19.

Table 19 (continued)

Table 20.

Table 20 (continued)

Table 21.

______

Blautia________ a preparation of a preparation of a preparation of a preparation of a preparation of Clostridiales glycan (as glycan (as glycan (as glycan (as glycan (as glycan (as glycan) Clostridium_____ describes herein describes in the describes in the describes in the Eubacterium document , for example, present present present Lachnospiraceae having any DP, document, by document, by document, by Roseburia_____ Db , link reason example, having example, having example, having Ruminococcus glycosidic alpha/beta, any DP, DB , any DP, DB, any DP, DB, number of bonds bond ratio bond ratio bond ratio glycosidic, regiochemistry alpha/beta glycosidic, alpha/beta glycosidic, alpha/beta glycosidic, bond and number of bonds number of bonds number of bonds, glycosidic, glycosidic, glycosidic, and other characteristics (for example regiochemistry, solubility, bonding and fermentability, viscosity, sweetness, etc.) bond , and other link, and other link, and others described herein characteristics (by characteristics (by characteristics (per document) that example, solubility, example, solubility, example, solubility, comprises glycans that fermentability, fermentability, fermentability, comprise a unit of viscosity, sweetness, viscosity, sweetness, viscosity, sweetness, mannose glycan, etc.) described in the etc.) described in the etc.) described in the optionally where present present present the preparation of glycan document) which document ) which document) which comprises any comprises glycans comprises glycans comprises glycans quantity of mannose of which comprises which comprises which comprises between 1% and 100%, glycan unit a unit of a unit of optionally in addition to rhamnose, glycan of fructose, fucose glycan, where the preparation of optionally in optionally in optionally in glycan comprises a where the where the where the

EXAMPLES

The invention is further illustrated by the following examples. The examples are provided for illustrative purposes only, and should not be construed as limiting the scope or content of the invention in any way. The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant RNA techniques and pharmacology, within the skill of the art. Such techniques are fully explained in the bibliography. See, for example, T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); Green & Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th edition (Cold Spring Harbor Laboratory Press, 2012); Colowick & Kaplan, Methods In Enzymology (Academic Press); Remington: The Science and Practice of Pharmacy, 22nd Edition (Pharmaceutical Press, 2012); Sundberg & Carey, Advanced Organic Chemistry: Parts A and B, 5th edition (Springer, 2007).

Example 1. Method for producing glycan polymers using a purified glycosidase enzyme Glycans can be generated by reverse hydrolysis of glycosidic bonds catalyzed by one or more partially or completely purified hydrolase or transferase enzymes, described herein, for example, tables 4, 22, 23 and 24 and, for example, encoded by SEQ ID Nos 1-124. The selected enzyme(s) may be any enzyme known to hydrolyze glycosidic bonds, including glycosylhydrolases, glycosyltransferases and polysaccharide lyases, including those that use non-activated sugar donors or activated sugar donors, including, but not limited to, sugar nucleotides and phosphoryl sugars. Ideally, the selected enzyme is isolated from or related to a glycosyl hydrolase of glycotaxons (e.g. class 1, class 2, class 3, class 4, class 5, class 6 or class 7) with a positive effect on the structure o host microbiome health (e.g. modulation of a metabolite, e.g. SCFA (e.g. butyrate, propionate), TMA/t Ma O, ammonia, uremic solute (e.g. indole, p-cresol), LPS or a bile acid (for example, a secondary bile acid).

The glycosyl donor may consist of an inactivated monomeric glycoside (e.g., glucose, galactose, glucuronic acid, etc.), an activated monomeric glycoside (e.g., phosphoglucose, lactose, maltobiose, UDP-glucose, 1-fluoroglucose, trichloroacetimidated glucose , para-nitrophenylglucose, hexeneuronic acid), or oligomeric or polymeric glycosides (for example, maltose, galactooligosaccharides).

The glycosyl acceptor may consist of any substrate compatible with glycosylhydrolases or transferases, including monomeric sugars (e.g., glucose, galactose, glucuronic acid), oligomers (e.g., lactose, maltobiose, galactooligosaccharides), or non-sugar substrates reported to They are compatible with other glycosides (e.g. sorbitol, glycerol). In some embodiments, the glycosyl acceptor and the glycosyl donor may be the same material, for example, maltobiose may be both donor and acceptor.

The enzyme of interest can be dissolved or suspended to a concentration of 1-50 U/ml in a biocompatible solvent including water; mixtures of water and a miscible solvent such as acetone, ethanol, isopropanol, polyethylene glycol, t-butanol or other notified solvents; or a pure organic solvent. Glycosyl donor and acceptor are added to the medium at a concentration between 5% and 50% by weight. The pH of the medium is adjusted to a range between 2.5 and 8.0 using a biocompatible buffer, for example, sodium acetate buffer, citric acid/disodium hydrogen phosphate buffer, phosphate buffer, etc. The reaction is then allowed to stir gently for 2-48 hours at a temperature compatible with the selected glycosidase enzyme molecule, typically 35°C to 90°C.

The conditions for synthesis using a glycosidase enzyme molecule that is isolated with minimal structural changes from its host or expressed from a recombinant system may more closely reflect native physiology, i.e., a pH between 5 and 7.5 and a temperature between 35 and 60 °C. Ideally, the conditions for synthesis using a glycosidase enzyme molecule that has been modified to improve its stability can further deviate from physiological conditions, for example a pH of between 3.5 and 8 and a temperature of between 35 and 70°. c. Changes in protein stability can be used to increase desirable properties, including synthesis yield, conversion rate, recyclability, or protein production yield.

Ideally, the conditions for synthesis using a glycosidase enzyme molecule that has been highly modified or isolated from Extremophilic bacteria evolved to withstand extreme temperature or pH conditions can deviate widely from physiological conditions, for example, a pH between 2.0 and 8 and a temperature of up to 90 °C.

In one embodiment, the selected substrate (Table 23, column E) was placed in a 20 ml scintillation vial. Mcllvaine buffer of specified pH (Table 23, column J) was added to the vial and the powder was dissolved by brief heating with a heat gun. After the buffered substrate solution was cooled to room temperature, the selected enzyme was added (Table 23, column H), the solution was shaken gently to mix, and the capped vial was placed in a water bath at the specified temperature (Table 23, column K), without stirring. After the specified time had elapsed (Table 23, column L), the vial was heated in a boiling water bath for 20 min to inactivate the enzyme. Samples were then transferred to 50 mL conical centrifuge tubes and diluted to −200 mg/mL. The diluted products were then purified by the methods described in Example 18. The isolated materials were characterized as described in Examples 11-15 and the data are presented in Table 23 (SEC) and Table 24 (HSQC-NMR). .

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Example 2. Generation of beta-galactooligosaccharides by means of beta-galactosidase and lactose

To a 0.05 M sodium acetate buffer, pH 5 at 60 °C, lactose was added to a concentration of 400 mg/ml. The reaction was stirred gently until the substrates were dissolved. A stock solution of beta-galactosidase (Megazyme, catalog no. E-BGLAN) was then added to the reaction to a final concentration of 10 U/ml. The reaction was maintained at 60 °C for 26 h in a covered water bath. When the reaction was considered complete, it was heated to 100 °C for 10 minutes to inactivate the enzyme, then diluted with water to a total carbohydrate concentration of 2% (Table 23, line 31). The products were then characterized, isolated and purified as described in examples 11-18 and shown in Figure 5. The isolated oligosaccharide has a molecular weight >20% DP>3, glycosidic stereochemistry >50% of beta-glucosides, a regiochemistry >10% of 1,4-glycosidic bonds and a constitution >50% of D-galactose.

Example 3. Generation of alpha-galactooligosaccharides by means of alpha-galactosidase and melibiose

To a 0.05 M sodium acetate buffer, pH 5 at 60 °C, melibiose was added to a concentration of 1000 mg/ml. The reaction was stirred gently until the substrates were dissolved. A stock solution of alpha-galactosidase (Megazyme, catalog no. E-AGLAN) was then added to the reaction to a final concentration of 10 U/ml. The reaction was maintained at 60 °C for 22 h in a covered water bath. When the reaction was considered complete, it was heated to 100 °C for 10 minutes to inactivate the enzyme, then diluted with water to a total carbohydrate concentration of 2% (Table 23, line 29). The products were then characterized, isolated and purified as described in Examples 11-18 (Figure 8). The isolated oligosaccharide has a molecular weight >20% DP>3, glycosidic stereochemistry >50% alpha-glucosides, regiochemistry >10% 1,4-glycosidic bonds, and constitution >50% D-galactose.

Example 4. Generation of beta-glucoigosaccharides by means of beta-glucosidase and cellobiose

Cellobiose was added to a 0.05 M sodium acetate buffer, pH 5 at 60 °C to a concentration of 200 mg/ml. The reaction was stirred gently until the substrates were dissolved. A stock solution of beta-glucosidase (Megazyme, catalog no. E-BGOSPC) was then added to the reaction to a final concentration of 10 U/ml. The reaction was maintained at 60 °C for 26 h in a covered water bath. When the reaction was considered complete, it was heated to 100 °C for 10 minutes to inactivate the enzyme and then diluted with water to a total carbohydrate concentration of 2% (Table 23, line 10). The products were then characterized, isolated and purified as described in Examples 11-18 (Figure 6). The isolated oligosaccharide has a molecular weight >10% DP>3, a glycosidic stereochemistry >50% beta-glucosides, a regiochemistry >10% 1,4-glycosidic bonds, and a constitution >90% D-glucose (Table 24 , line 10).

Example 5. Generation of alpha-glucoigosaccharides by means of alpha-glucosidase and maltose

To a 0.05 M sodium maleate buffer, pH 6.5 at 55 °C, maltose was added to a concentration of 1000 mg/ml. The reaction was stirred gently until the substrates were dissolved. A stock solution of alpha-glucosidase (Megazyme, catalog no. E-TSAGL) was then added to the reaction to a final concentration of 10 U/ml. The reaction was maintained at 55 °C for 24 h in a covered water bath. When the reaction was considered complete, it was heated to 100 °C for 10 minutes to inactivate the enzyme, then diluted with water to a total carbohydrate concentration of 2% (Table 23, line 27). The products were then characterized, isolated and purified as described in Examples 11-18 (Figure 7A, B). The isolated oligosaccharide has a molecular weight >15% DP>3, a glycosidic stereochemistry >50% alpha-glucosides, a regiochemistry >10% 1,4-glycosidic bonds, and a constitution >90% D-glucose (Table 24 , line 11).

Example 6. Selective generation of oligosaccharides by treating substrate mixtures with a selective glycosidase

Oligosaccharides with mixed compositions (including mixtures of stereochemistry, regiochemistry, and monomeric composition) are generated by treating a mixture of substrates with a glycosidase that is selective for one or more of the substrates under the conditions described in Example 1. In this example, the substrates glycosidase susceptible serve as glycosidic bond donors and substrates that are not glycosidase susceptible only serve as glycosidic bond acceptors. By mixing different combinations of substrates with differently selective glycosidases, different combinations of oligosaccharides can be generated (Figure 12).

In one embodiment, a 1:2 w/w mixture of cellobiose and lactose was added to a 0.05 M sodium acetate buffer, pH 5 at 60 °C to a final concentration of 600 mg/ml. The reaction was stirred gently until the substrates were dissolved. A stock solution of beta-glucosidase (Megazyme, catalog no. E-BGOSPC) was then added to the reaction to a final concentration of 10 U/ml. The reaction was maintained at 60 °C for 26 h in a covered water bath. When the reaction was considered complete, it was heated to 100 °C for 10 min to inactivate the enzyme and then diluted with water to a total carbohydrate concentration of 2%. The products were then characterized, isolated and purified as described in Examples 11-18 (Figure 14). The isolated oligosaccharide has a molecular weight >10% DP>3, a glycosidic stereochemistry >50% beta-glucosides, a regiochemistry >10% 1,4-glycosidic bonds, and a constitution >50% D-glucose and <50 % D-galactose.

In a second embodiment, a 1:2 w/w mixture of cellobiose and lactose was added to a 0.05 M sodium acetate buffer, pH 5 at 60 °C, to a final concentration of 600 mg/ml. The reaction was stirred gently until the substrates were dissolved. A stock solution of beta-galactosidase (Megazyme, catalog no. E-BGLAN) was then added to the reaction to a final concentration of 10 U/ml. The reaction was maintained at 60 °C for 26 h in a covered water bath. When the reaction was considered complete, it was heated to 100 °C for 10 min to inactivate the enzyme and then diluted with water to a total carbohydrate concentration of 2%. The products were then characterized, isolated and purified as described in Examples 11-18 (Figure 13). The isolated oligosaccharide has a molecular weight >20% DP>3, a glycosidic stereochemistry >50% beta-glucosides, a regiochemistry >10% 1,4-glycosidic bonds, and a constitution of <50% D-glucose and >50 % D-galactose.

In other embodiments, the selected substrate mixture may differ by the constituent monomer (e.g., maltose and melibiose or L-arabinbiose and D-arabinobose), by glycosidic stereochemistry (e.g., maltose and cellobiose), by regiochemistry (e.g. for example, lactose and allolactose), by activation strategy (for example, 1-(para-nitrophenyl)-D-glucose and lactose), or by any combination of these or other factors that differentiate glycosidic enzyme substrates. Selected glycosidases may differ based on substrate selectivity (e.g., glucosidase vs. galactosidase), glycosidic stereochemistry (e.g., alpha-glucosidase vs. beta-glucosidase), regiochemistry (e.g., 1,6-glucosidase vs. 1 ,3-glucosidase), chain position (e.g., endoglycosidase vs. exoglycosidase; selective reducing end vs. selective non-reducing end), or any combination of these or other factors that differentiate glycosidic enzymes.

Example 7. Generation of oligosaccharides from monomeric sugars using a mixed organic/aqueous solvent system

The glycans described in Example 1 can be generated from monomeric sugars through a thermodynamic reverse hydrolysis event. Sugar hydrolases establish a thermodynamic equilibrium in which monomeric sugars are in constant exchange with higher order oligosaccharides with the dominant direction of the reaction dictated by the water activity of the environment. The high concentration of water in relation to the substrate found under physiological conditions means that these enzymes have a mainly hydrolytic action. Under certain laboratory conditions, the dominant direction of the reaction can be altered to favor the formation of glycosidic bonds that allow the formation of oligosaccharides from monomeric constituents. Glucose was dissolved in 0.1 M sodium acetate buffer, pH 5.2, at a concentration of 10% (w/w). After the substrates were dissolved, four volumes of one of the following organic solvents were added to the aqueous portion: tert-butanol, diethylene glycol dimethyl ether (DEGD), tetraethylene glycol dimethyl ether (TEGD), or trimethyl phosphate (TMP). Beta-glucosidase stock solution was added to the reaction to a concentration of 10 U/ml. The reaction was heated to 50 °C in a covered water bath and monitored over a period of 10 days. Once the reaction was considered complete, the reaction was heated at 100 °C for 10 min to inactivate the enzymes. The reaction was then centrifuged in a benchtop centrifuge to collect the precipitated glycans, and the supernatant was removed by decantation. The products were then characterized and purified as described in examples 11-18. A fluorophore-assisted carbohydrate electrophoresis experiment demonstrating the presence of DP>1 glycans is shown in Figure 9.

Example 8. Changing product distributions by adding reaction modifiers

Other modifiers can be added to the reaction to control the reaction rate, total material yield, conversion efficiency, or structural distribution of the resulting glycan. In one embodiment, monomeric D-galactose was added to the reaction described in Example 12. Comparison of the product profile over 24 hours showed that galactose both slowed the rate of oligosaccharide formation and shifted the product distribution toward a higher proportion. of oligosaccharides (figure 10).

To a 0.05 M sodium acetate buffer, pH 5 at 60 °C, lactose was added to a concentration of 400 mg/ml. The lactose was stirred gently until it dissolved. D-galactose was then added to the reaction to a final concentration of 115 mg/ml and gently stirred until dissolved. A stock solution of beta-galactosidase (Megazyme, catalog no. E-BGLAN) was then added to the reaction to a final concentration of 10 U/ml. The reaction was maintained at 60 °C for 24 h in a covered water bath. When the reaction was considered complete, it was heated to 100 °C for 10 min to inactivate the enzyme and then diluted with water to a total carbohydrate concentration of 2%. The products were then characterized, isolated and purified as described in examples 6-8.

Example 9. Method for producing glycans by means of reverse hydrolysis using bacterial culture isolate

The glycans described, for example, in Example 1, can be generated by reverse hydrolysis of glycosidic bonds catalyzed by an isolated bacterial preparation that includes one or more of the categories of viable whole cells, non-viable whole cells, partial cells or cellular components, cell culture supernatants or other portions of bacterial media containing glycoside hydrolases. In this experiment, first a bacterial strain, a combination of strains or an entire bacterial community is grown on suitable media and under suitable conditions. The culture is then harvested using conventional techniques and then the isolated culture is subjected to the reverse hydrolysis conditions described in Example 1 to generate a glycan that is adapted to that strain, combination or community.

The strain, combination or community of strains can be grown using any of the well-known media described, for example, in Martens, EC;et al.Cell Host & Microbe, 2008, 4, 447.; Romano, KA;et al.mBio, 2015, 6, e02481-14.; Atlas, R. M., Handbook of Microbiological Media, 4th ed., 2010.; or other published literature that selects culture conditions optimized for the strain or strains of interest. Once the culture has reached a suitable density, the culture is collected from the medium by any suitable method including filtration, centrifugation or lyophilization.

If desired, isolated cells are lysed after collection by bead beating, lyophilization, or other technique to produce a mixture of nonviable cellular matter containing natively expressed endocellular or membrane-bound glycosyl hydrolases.

If desired, the culture supernatant is isolated and lyophilized independently of cellular matter to produce a mixture of natively expressed exocellular glycosyl hydrolases.

If desired, the combined cells and culture supernatant are lysed and lyophilized together to produce a mixture of all expressed glycosyl hydrolases.

The isolated glycosyl hydrolases described are then used to generate glycans, for example, as described in example 1 using the isolated glycosyl hydrolases instead of the purified enzymes. The conditions described in Example 1 can be further modified by the addition of cell permeabilizing agents, including DMSO or toluene, to increase the accessibility of the expressed enzymes. The activity of the isolated solids can be measured using any suitable technique, for example, described in Goulas, et al. International Dairy Journal 17 (2007) 648-656. When applied to glycans generated using this method, the purifications described in Examples 16-18 can be altered to remove residual cellular material by any well-known method including centrifugation, sterile filtration, and gel filtration chromatography. The glycans can then be used to increase the growth of particular strains, combinations or communities of strains, for example, in the intestine, for example, upon administration of an effective amount to a human subject.

In a further embodiment, a biomass pellet containing -400 mg of Bifidobacterium longum (BLO.16) grown in a medium containing glucose as the sole carbohydrate source was washed with 1 ml of 0.01 XPBS six times to remove any residue of the culture medium. For each wash, the pellet was resuspended by vortexing and then collected by centrifugation at 10,000 g for 3 minutes. Once washing was completed, the biomass was resuspended in water at -100 mg/ml and diluted with 16 ml of 0.01 XPBS and 40 |il of toluene for 1 h at room temperature to increase cell membrane permeability. The cell pellet was collected by centrifugation at 10,000 g for 10 minutes and the supernatant was discarded. The pellet was then washed four times with 20 ml of 0.01 XPBS in the same manner. After that, the biomass was resuspended in 4 mL of water and lyophilized for enzymatic synthesis purposes.

After biomass preparation, 250 mg of maltose was weighed into a 1.5 ml microcentrifuge tube and dissolved in 250 μl of 0.05 M acetate buffer, pH 5, with brief heating in a water bath. at 40°C. The freeze-dried biomass was added to the maltose solution. Reactions were maintained at 40 °C for 3 days and oligomer production was monitored. At each time interval, a 20 µl portion of a sample was heated in a boiling water bath for 10 minutes to inactivate the enzyme. This portion was then diluted to -20 mg/ml with water and filtered through 0.2 μm to check the reaction progress by SEC or TLC.

Example 10. Method for inducing hydrolasse expression by pretreatment of the bacterial culture with defined glycan

The method for producing glycans by an isolated bacterial preparation described in Example 3 can be further modified by inducing the expression of specific glycosyl hydrolases in the culture growth stage in order to change the expression levels of the enzyme. In this example, the bacterial culture conditions described in Example 8 can be modified by the addition of a glycan (e.g., from a previous synthesis), glycoside (e.g., alpha-1,6-mannobiose), oligosaccharide (e.g. , lactose, GOS) or fiber (e.g., cellulose, inulin, pectin) instead of other carbon sources in order to induce the crop to express glycosyl hydrolases specific for the metabolism of that glycan, glucoside, oligosaccharide or fiber as a substrate . In this way, the enzymatic activity of the isolated bacterial preparation can be adjusted and enhanced to improve the production of the desired glycan. For example, a cell isolate that favors the production of alpha-1,6-glucose linkages is generated by growing the desired strain on oligosaccharides containing predominantly alpha-1,6-glucose linkages such as isomaltose. The culture is established with isomaltose as the sole carbon source until the strain reaches a growth rate that suggests upregulation of enzymes capable of digesting isomaltose. Isolates from this culture are enriched in glycosidases selective for the hydrolysis of alpha-1,6-glucose. This enriched isolate is then used to produce glycans via reverse hydrolysis. The glycan can be administered to a subject, for example, to the intestine, to selectively stimulate the growth of a bacterial taxon rich in alpha-1,6-glucosidase.

In a second embodiment, the biomass described in example 9 was grown in a medium containing melibiose (galactose-alpha-1,6-glucose) as the sole source of carbohydrates instead of glucose, resulting in a biomass with glucosyl downregulated hydrolases and upregulated galactosylhydrolases. The procedure described in example 9 was then modified by replacing the maltose with 100 mg of melibiose dissolved in 250 µl of citric/phosphate buffer pH 5.5. The procedure was then followed identically to create alpha-galactose oligomers instead of alpha-glucose as described in Example 9. Example 11. Glycan Preparations

To a round bottom flask equipped with an overhead stirrer and a jacketed short path condenser was added one or more mono- or disaccharides together with 3-20% by dry weight of one or more of the catalysts, e.g. containing acids, ionic, ionic/acids such as, for example, those described in US Patent Nos. 9,079,171 and WO 2016/007778. Water or another compatible solvent (zero to 10 equivalents) was added to the dry mixture and the suspension was combined at approximately 100 rpm using a paddle sized to match as closely as possible the contours of the selected round bottom flask. The mixture was then heated to 80-185 °C. Once the solids reached a molten state, the vessel was placed under a vacuum pressure of 10-1000 mbar. The reaction was stirred for 30 minutes to 8 hours, constantly removing water from the reaction. The progress of the reaction was monitored by HPLC. When sufficient oligomerization had occurred, the stirrer was turned off, the reaction was cooled to room temperature and vented to atmospheric pressure, and the product, either as a solid or as a syrup, was dissolved in a volume of water sufficient to create a solution of approximately 50 Brix (grams of sugar per 100 g of solution). After complete dissolution, the solid catalyst was removed by filtration and the oligomer solution was concentrated to approximately 50-75 Brix by rotary evaporation. In cases where an organic solvent has been used, water-immiscible solvents can be removed by two-phase extraction and water-miscible solvents can be removed by rotary evaporation concomitant with the concentration step.

Among others, the following glycans were prepared in multiple batches and tested in various assays described herein:

Single glycan unit (homoglycans): ara100, fru100, gal100, galA100, glcNac100, glu100, gluA100, Lglu100, man100, rha100, xyl10.

Two glycan units (heteroglycans): Ara60Xyl40, Ara80Xyl20, Gal20Ara80, Gal20Xyl80, Gal40Ara60, Gal40Man60, Gal40Xyl60, Gal57Glu43, Gal60Ara40, Gal60Man40, Gal60Xyl40, Gal80Ara20, Gal80Man20, Gal80Xyl20, Glu2 0Ara80, Glu20Xyl80, Glu40Ara60, Glu40Gal60, Glu40Xyl60, Glu50Gal50, Glu50Lglu50, Glu60Ara40, Glu60Gal20Man20, Glu60Gal40, Glu60Man40, Glu60Xyl40, Glu66Fru33, Glu75Gala25, Glu75GluA25, Glu75GluN25, Glu80Ara20, Glu80Gal20, Glu80Lglu20, Glu80Man20, Glu80Xyl20, Man20Ara80, Man20Xyl80, Man40Ara60, Man40Xyl60, Man60Ara40, Man60Glu40, Man60Xyl40, Man75Gal25, Man80Ara20, Man80Gal20, Man80Glu21, Man80Xyl20, Xyl60Ara40, Xyl75Ara25, Xyl80Ara20, and the hybrid glycans glu90sor10 and glu90gly10.

Three glycan units (heteroglycans): Gal5Xyl5Ara90, Gal5Xyl90Ara5, Gal10Xyl10Ara80, Gal10Xyl45Ara45, Gal10Xy180Ara10, Gal20Xyl20Ara60, Gal20Xyl40Ara40, Gal20Xyl60Ara20, Gal30Xyl30Ara40, 0Ara30, Gal33Man33Ara33, Gal33Man33Xyl33, Gal33Xyl33Ara33, Gal45Xyl10Ara45, Gal45Xyl45Ara10, Gal50Glu25Fru25, Gal40Xyl20Ara40, Gal40Xyl30Ara30, Gal40Xyl40Ara20, Gal60Xyl20Ara20, Gal80Xyl10Ara10, Gal90Xyl5Ara5, Glu5Gal5Man90, Glu5Gal90Man5, Glu5Xyl5Ara90, Glu5Xyl90Ara5, Glu10Gal10Man80, Glu10Gal45Man45, Glu10Gal80Man10, Glu10Xyl10Ara80, Glu10Xyl45Ara45, Glu10Xyl80Ara10, 0Man60, Glu20Gal40Man40, Glu20Gal60Man20, Glu20Gal80, Glu20Xyl20Ara60 Glu20Xyl40Ara40, Glu20Xyl60Ara20, Glu30Gal30Man40, Glu30Gal40Man30, Glu30Xyl30Ara40, Glu30Xyl40Ara30, ra33, Glu33Gal33Fuc33, Glu33Gal33Man33, Glu33Gal33Xyl33 , Glu33Man33Ara33 Glu33Man33Xyl33, Glu33Xyl33Ara33, Glu40Gal20Man40, Glu40Gal30Man30, Glu40Gal40Man20 Glu40Xyl20Ara40, Glu40Xyl30Ara30, Glu40Xyl40Ara20, Glu45Gal10Man45, Glu45Gal45Man1 0 Glu45Xyl10Ara45, Glu45Xyl45Ara10, Glu60Xyl20Ara20, Glu75GluNAc25, Glu80Gal10Man10 Glu80Xyl10Ara10, Glu90Gal5Man5, Glu90Xyl5Ara5, Man33Xyl33Ara33, Man52Glu29Gal19. Four glycan units (heteroglycans): Gal25Man25Xyl25Ara25, Glu25Gal25Man25Ara25, Glu25Gal25Man25Xyl25, Glu25Gal25Xyl25Ara25, Glu25Man25Xyl25Ara25.

Five glycan units (heteroglycans): Glu20Gal20Man20Xyl20Ara20.

Glycans are described by a three- to six-letter code that represents the monomeric sugar component followed by a number in one hundred that reflects the percentage of the material that makes up the monomer. Therefore, 'glu100' is attributed to a glycan generated from a 100% contribution of D-glucose (glycan unit) and 'glu50gal50' is attributed to a glycan generated from a 50% contribution of D- glucose and 50% of D-galactose (glycan units) or, alternatively from a contribution of lactose dimer (glycan unit). As used herein: xyl = D-xylose; ara = L-arabinose; gal = D-galactose; glu = D-glucose; rha = L-rhamnose; fuc = L-fucose; man = D-mannose; sor = D-sorbitol; gly = D-glycerol; neu = NAcneuraminic acid; Lglu = L-glucose; gluA = D-glucuronic acid; gluN = D-glucosamine; gluNAc = N-acetyl-D-glucosamine; galA = D-galacturonic acid. 3-Bn = benzyl; 3-Obn = 3-benzyloxy; 6-TBDPS = 6-tert-butyldiphenylsilyl; galnac = N-acetyl galactosamine; rib = D-ribose; Sor = sorbitol.

Example 12. Purification

Oligo- and polysaccharides were dissolved in deionized water to a final concentration of 25-50 Brix. The material was then exposed to at least 2 mass equivalents of Dowex Monosphere 88 ion exchange resin. Exposure can occur by shaking a flask at 120-170 rpm or by filtration through a wet slurry packed column provided the time of residence is sufficient for the solution to reach a final pH of between 3 and 5. The oligomer solution was isolated by filtration (as in the case of stirred reactions) or elution (as in the case of column filtration) and the process was repeated with Dowex Monosphere 77 ion exchange resin in an analogous manner until the pH of the solution was above 5.5. Finally, the solution was exposed to Dowex Optipore s D-2 adsorbent decolorizing resin until the solution was sufficiently clarified and filtered through a 0.2 micrometer filter to remove residual resin and resin fines. The final solution was then concentrated to 50-85 Brix by rotary evaporation or to a solid by lyophilization.

Example 13. Small-scale high-throughput preparation

Oligomers and polymers were synthesized in parallel in 24-, 48-, or 96-well plates or in similarly sized arrays of 1 dram vials housed in aluminum heating blocks. In this example, all liquid transfers were handled by programmable robot or manually using calibrated pipettes. To each vial or well was added 20-100% by dry weight of one or more catalysts, for example, acid-containing catalysts, ionic catalysts, ionic catalysts/acid-containing catalysts such as, for example, those described in the patent US Patent No. 9,079,171 and WO 2016/007778. The heating plate or block was placed uncovered in a vacuum oven heated to 50 to 150 °C under a vacuum of 10-800 mbar. The furnace vacuum pump was protected by a two-stage condenser consisting of a recirculating chiller trap followed by a dry ice/acetone trap. The plates or blocks are heated for 30 minutes to 6 hours at elevated temperature and reduced pressure without agitation. After a preset period of time, the oven was vented to atmospheric pressure, the plates or blocks were cooled to room temperature, and each well or vial was diluted to approximately 50 Brix with deionized water. The solid phase extraction steps described in Example 12 were performed by elution through sequential wet-packed columns in which the eluent from each column flows immediately to the top of the next column at a rate between 2 and 6 bed volumes/hour using a peristaltic pump or other suitable small pump. The column stack was then rinsed with deionized water, and the combined effluents were concentrated by freeze-drying to isolate solid powders with a residual water content of 1–10% by mass.

Example 14. Elimination of low molecular weight species

Oligomers or polymers were modified to remove low molecular weight species.

In one embodiment, the separation was achieved by osmotic separation. Approximately 45 cm of 1.0 kD MWCO Biotech CE dialysis tubing (31 mm flat width) from Spectrum Labs was placed in deionized water and soaked for 10 minutes, then one end was sealed with a dialysis tubing clamp. . A 25 Brix solution of 8 grams of dry oligosaccharide was filter sterilized and sealed in the tube with a second clamp along with a few ml of air to allow the tube to float. The filled tube was then placed in a 3-gallon tank of deionized water that was shaken vigorously enough to induce a slow swirl of the sealed tubes. After 8 hours, the water in the tank was replaced and the tube was allowed to agitate for an additional 16 hours. Once dialysis was completed and the material had greater than 95% DP2+ yield and greater than 90% DP3+ yield, the diluted solution was filter sterilized and concentrated in vacuo to a final concentration of approximately 65 Brix or It was lyophilized until obtaining a solid with a residual humidity of between 1 and 10%.

In a second embodiment, separation was achieved by tangential flow filtration (TFF). In this case, 100 mL of a 25 Brix glycan sample dissolved in filter-sterilized deionized water was placed in the feed bottle of a Spectrum Labs KrosFlo Research IIi TFF system that was prepared according to the manufacturer's recommendation. The sample was then subjected to diafiltration through a 1 kD MidiKros mPES hollow fiber filter at a transmembrane pressure of 25 psig. HPLC samples of the raw material taken every 0.5 volume of diafiltration were used to determine when the material had a DP2+ yield greater than 95% and a DP3+ yield greater than 90%, at which time the solution was sterilized by filtration and concentrated in vacuo to a 65 Brix syrup or lyophilized to a solid with a residual water content of 1-10% by mass.

In a third embodiment, the separation was achieved by ethanol precipitation. In this case, 100 ml of a 25 Brix glycan sample was poured into a vigorously shaken beaker containing 900 ml of pure USP grade ethanol at a rate of no more than 10 ml/minute. Once the addition was complete, the precipitated solids were stirred for an additional 15 minutes at room temperature or slightly below. The precipitated solids were isolated by filtration through a fine frit sintered glass funnel under a nitrogen atmosphere to prevent hydration and gumming. The solids were rinsed once with ethanol, then dissolved in water to a final concentration of 25 Brix and reconcentrated to >65 Brix. This syrup was then diluted again to 25 Brix and concentrated once again to ensure removal of residual ethanol.

Example 15. Methods for analyzing preparations.

Concentration measurement by liquid refractometry

This experiment was designed to quantify the amount of glycan in any given aqueous solution. A Mettler-Toledo Refracto 30GS portable sugar refractometer was calibrated using high purity reverse osmosis deionized water. Several drops of the glycan solution were filtered through a 0.2 micrometer syringe filter directly onto the refractometer lens. The measurement was taken at room temperature and reported as Brix. Glycans were routinely concentrated to 50, 60, 70, or 75 Brix without obvious solidification or crystallization at 23 °C. Brix can then be converted to solubility assuming a specific gravity of water equal to 1.0 g/ml. Therefore, 75 Brix (100 grams of solution consisting of 75 grams of glycan and 25 grams of water) is equivalent to an aqueous solubility of 3.0 g/ml. For comparison, Sigma-Aldrich reports the aqueous solubility of D-glucose to be 0.909 g/ml (48 Brix) at 25°C.

Monomeric composition by hydrolysis and GC-MS.

This experiment was designed to quantify the ratio of monomer content within a given oligosaccharide. Glycosyl composition analysis was performed by combined gas chromatography/mass spectrometry (GC/MS) of per-O-trimethylsilyl (TMS) derivatives of monosaccharide methylglycosides produced from the sample by acid methanolysis such as previously described by Santanderet al.(2013) Microbiology 159:1471. Between 100 and 200 μg of sample were lyophilized in a suitable test tube. Inositol (20 μg) was added to the sample as an internal standard and then the sample was heated to 80 °C in 1 M HCl/methanol for 18 hours. The resulting monosaccharides were then re-acetylated using pyridine and acetic anhydride in MeOH, and per-O-trimethylsilylated with Tri-Sil (Pierce) at 80 °C for 30 min. GC/MS analysis of TMS methyl glycosides was performed on an Agilent 7890A GC instrument interfaced to an MSD 5975C instrument, using a Supelco Equity-1 fused silica capillary column (30 m x 0.25 mm ID). Each peak was assigned to a sugar component based on comparison with known standards, and integration of the respective peaks allowed a clean calculation of the relative percentage of monomers within an exemplified glycan. In all of the glycans listed, conditions can be routinely identified in which the monomer composition of a given oligosaccharide matched the input ratio within the experimental error and the output composition matched the input composition within the precision of the measurement.

Molecular weight distribution by size exclusion chromatography (SEC)

This experiment was designed to quantify the molecular weight distribution within a given oligosaccharide. Measurement was performed by HPLC using the method described in United States Pharmacopeia Monograph, 38(6) Revision in Progress: Heparin Sodium (USP37-NF32). Separations were achieved on an Agilent 1200 HPLC system via an overlay GE column 12 using 50 mM ammonium acetate as eluent at a flow rate of 1.0 ml/min and an ELSD detector. The column temperature was set to 30 °C and dextran (1 kD, 5 kD, 10 kD wt) was used to draw a standard curve. A 2 mg/ml solution of the samples was prepared and passed through a 0.45 µm spin filter, followed by injections of 40 µl into the HPLC. A third-order polynomial curve was constructed based on the log molecular weights and elution volumes of the listed standards. The weight average molecular weight (Mw), number average molecular weight (Mn) and polydispersity index (PDI) were calculated for the sample by comparison with the standard curve. Figure 1 shows the curve generated during SEC evaluation of a glu100 sample in which the average molecular weight was determined to be 1212 g/mol or approximately DP7. The upper end of the molecular weight of the material defined by the curve point at the 10% maximum absorption leading the curve was determined to be 4559 g/mol or approximately DP28. The lower end of the molecular weight of the material defined by the 10% maximum absorption behind the curve was determined to be 200 g/mol or approximately DP1. A similar analysis of a glu50gal50 sample showed a MW, high mass, and low mass of 1195 g/mol (~DP7), 4331 g/mol (-DP27), and 221 g/mol (~DP1), respectively.

Molecular weight distribution by ion affinity chromatography (IAC)

The proportion of glycan with DP greater than or equal to 2 (DP2+) and 3 (DP3+) can be measured by ion affinity chromatography. A glycan sample was diluted to 50-100 mg/ml and 10 µl of this solution was injected into an Agilent 1260 BioPure HPLC instrument equipped with a BioRad Aminex HPX-42A 7.8 x 300 mm column and a detector. RI. Using pure HPLC grade water as eluent, the sample was eluted at 0.6 ml/min through an 80 °C column and an RI detector maintained at 50 °C. Peaks representing DP1-6 are assigned by comparison with reference standards and integrated using Agilent ChemStation software. The peaks are typically integrated as DP1, DP2, DP3, DP4-7, and DP8+. The DP that can be achieved by the reaction described in Example 11 varies from one monomer to another, although it is constant between batches if the procedure is followed. For example, in 17 batches of glu100, DP2+ values ranged from 77-93% and DP3+ values ranged from 80-90%. In contrast, in 6 lots of ara100, DP2+ values ranged from 63-78% and DP3+ values ranged from 48-71%. The monomer mixtures behaved as averages of the individual components.

Alpha/beta distribution by 2D NMR

This experiment was designed to quantify the ratio of alpha and beta glycosidic bonds within a given sample using two-dimensional NMR. Approximately 150 mg of 65 Brix oligosaccharide solution was dried to a stable mass in a vacuum oven at 45-95 °C under a pressure of 400 mbar. The sample was subjected to two cycles of dissolution in D2O and drying to remove residual H2O. Once dry, the sample was dissolved in 750 |il D2O with 0.1% acetone, placed in a 3 mm NMR tube and analyzed on a Bruker Avance-III instrument operating at 500.13 MHz 1H (125.77 MHz 13 C) equipped with a Bruker BBFO probe operating at 21.1 °C. The sample was analyzed using a heteroatomic single quantum coherence (HSQC) pulse sequence using the standard Bruker pulse sequence. Anomeric protons between 4-6 ppm (1H) and 80-120 ppm (13C) were assigned by analogy to glucose as reported in Roslundet al.(2008) Carbohydrate Res. 343:101-112. Spectra were referenced to the internal acetone signal: 1H - 2.22 ppm; 13°C - 30.8 ppm. The isomers were quantified by integrating their respective peaks using the MNova software package from Mestrelab Research (Santiago de Compostela, Spain). Figure 2 shows the anomeric region of a representative spectrum. More than 300 samples have been analyzed in this way and Table 29 lists the distribution in a sample of monomer combinations showing that the alpha/beta ratio is as high as 4:1 as in the case of rha100 and as low as 1 :1 as in the case of glu50gal50.

Table 29: Distribution of alpha and beta bonds across batches and glycan types

This experiment was designed to identify the composition of a glycan by 2D NMR identification of the constituent monomers. Approximately 150 mg of 65 Brix oligosaccharide solution was dried to a stable mass in a vacuum oven at 45-95 °C under a pressure of 400 mbar. The sample was subjected to two cycles of dissolution in D2O and drying to remove residual H2O. Once dry, the sample was dissolved in 750 |il D2O with 0.1% acetone, placed in a 3 mm NMR tube and analyzed on a Bruker Avance-III instrument operating at 500.13 MHz 1H (125.77 MHz 13 C) equipped with a Bruker BBFO probe operating at 70 °C. The sample was analyzed using a heteroatomic single quantum coherence (HSQC) pulse sequence using the standard Bruker pulse sequence. The anomeric region of each glycan spectrum derived from a single sugar monomer was then examined for peaks representing specific glycosidic bonds characteristic of that monomer. For any given glycan, HSQC spectra allow the identification of peaks that are unique to the regio- and stereochemical arrangement of the bonds. For example, Figure 5 shows a partial mapping of the spectra of a glu100 preparation demonstrating how these peaks can be used to identify specific glycosidic regio- and stereochemistries. Due to the spin-isolated nature of the individual carbohydrate rings within polysaccharides, the HSQC spectra of a glycan with more than one monomer are predicted to be represented by the sum of the HSQC peaks of each of its constituent sugars. . Therefore, each constituent monomer has unique HSQC peaks that will appear on any glycan containing that monomer independently of other constituent monomers, and furthermore, the monomers used to synthesize a glycan can be determined by identifying the unique fingerprint peaks for each constituent monomer. For example, Figure 3B shows that the HSQC spectrum of glu50gal50 is a hybrid of the spectra of glu100 (Figure 3A) and gal100 (Figure 3C). Table 30 lists the fingerprint peaks for selected glycan units.

Table 30: Diagnostic HSQC peaks for each component sugar.

At least 5 peaks appeared for each glycan unit used as starting material in the synthesis of glycans containing 3 or fewer distinct glycan units. HSQC spectra of glycans containing 4 or more distinct glycan units have at least 4 peaks for each constituent glycan unit.

Figures 6A and 6B show the HSQC spectra for man 100 and xyl 100, respectively.

Analysis of glycosidic linkages

This experiment was designed to quantify the distribution of glycosidic regioisomers (branching) within a given oligosaccharide. For glycosyl linkage analysis, samples were permethylated, depolymerized, reduced, and acetylated; and the resulting partially methylated alditol acetates (PMAA) were analyzed by gas chromatography-mass spectrometry (GC-MS) as described by Heisset al(2009) Carbohydr. Res. 344:915. The samples were suspended in 200 µl of dimethyl sulfoxide and allowed to stir for 1 day. Permethylation was affected by two rounds of treatment with sodium hydroxide (15 min) and methyl iodide (45 min). The aqueous solution was hydrolyzed by adding 2 M trifluoroacetic acid and heating to 121 °C for 2 hours. The solids were isolated in vacuo and acetylated in acetic acid/trifluoroacetic acid. The resulting PMAAs were analyzed on an Agilent 7890A GC instrument connected to an MSD 5975C instrument (mass selective detector, electron impact ionization mode); The separation was performed on a 30 m Supelco SP-2331 bonded-phase fused silica capillary column. Figure 4 shows three representative GC spectra from this analysis. These analyzes show that the glycans had at least 0.1%, 0.2%, 0.5%, 1%, 2%, 5%, 10% or more of the 1,2-glycoside linkage type, e.g. , ara100 = 3.8%, gal 100 = 7.2%; at least 0.1%, 0.2%, 0.5%, 1%, 2%, 5%, 10% or more of the 1,3-glycoside bond type, e.g. 3-bn-glu100 = 1.7%, glu50gal50 = 10.4%; at least 0.1%, 0.2%, 0.5%, 1%, 2%, 5%, 10% or more of the 1,4-glycoside bond type, e.g. glu50gal50 = 5.9% , gal33man33ara33 = 10.1%; and at least 0.1%, 0.2%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25% or more of the 1,6-glycosidic bond type , for example, gal33man33ara33 = 13.4%, glu100 = 25.4%. The materials also contained at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50% or more of branched linkage types (including, but not limited to, 1,3, 6-; 1,4,6-; or 1,2,4-glycosides, for example, table 31), a degree of branching (DB) of at least 0.05. The degree of branching is defined as the average number of branched monomers relative to the total number of monomer units. For example, a glu100 glycan polymer in which 20% of the glucose monomer units contain glycosidic linkages to three or more glucose monomers would have a DB of 0.20. Glycans also have approximately 3-12% of the monomeric units in the form of furanose. A glycan originating from a single monomer consisted of at least 12 distinct non-terminal substitution patterns. A glycan originating from two monomers consisted of at least 18 distinct non-terminal substitution patterns, for example, glu-1,2-glu; glu-1,2-gal; gal-1,2-glu; gal-1,2-gal; glu-1,2(glu),6-glu; glu-1,3-glu; glu-1,3-gal; etc A glycan originating from three or more monomers consisted of at least 24 distinct non-terminal substitution patterns.

Different preparations characterized as described herein.

Fluorescence-assisted carbohydrate electrophoresis (FACE)

This experiment was designed to quantify the molecular weight of glycans within a given oligosaccharide in cases where the background signal (e.g., bacterial culture material, residual enzymes, buffers, etc.) interferes with detection techniques. s>E<c>traditional. The selected glycan was labeled with a polyanionic dye such as 8-aminopyren-1,3,6-trisulfonic acid (APTS) or 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and separated by gel electrophoresis. The selected glycans were diluted to 100 mg/ml in water and 5 |il of this solution was diluted to 1.00 ml to obtain a final solution concentration of 0.5 |ig/|il. To 10 nmol of glycan dried under vacuum for 3 h, 2 µl of 0.1 M APTS or ANTS, 2 µl of NaCNBH31 M in THF and 2 µl of 1.7 M citric acid were added and the mixture was kept at 70 °C for 2 h. The labeled solutions were diluted with 94 µl of water and subsequently 4 µl of this labeled glycan solution were mixed with 1 µl of 40% glycerol in water and loaded onto a precast 20% polyacrylamide gel (Life- Technologies). Electrophoresis was performed for 5 min at 100 V and then at 400 V for 40 min at 4 °C. Maltodextrin and pullulan 12,000 were treated in the same manner and used as standards in each gel. Gels were visualized with a Bio-Rad Gel Doc EZ Imager instrument using the Bio-Rad Image Lab 3.0 software package. Images captured with Bio-Rad software were processed using GE ImageQuant TL 8.1 software.

The reaction conditions can be adjusted as described herein to achieve a desired average degree of polymerization, degree of branching and distribution of glycosidic bonds to generate glycan preparations that are effective in modulating the production or level of a metabolite when administered, for example, to the intestine of a subject, and are substrates of a glycosidic enzyme (for example, a glycosidase enzyme that is present in a human intestinal microbe) and, if desired, the human intestinal microbe is one of the glycotaxons selected from class 1, class 2, class 3, class 4, class 5, class 6 or class 7, as described herein.

Example 16. Purification of glycan polymer preparations by ion exchange chromatography

If desired, any glycan generated as described herein is purified by ion exchange chromatography. Oligo- and polysaccharides were synthesized and dissolved in deionized water to a final concentration of 25-50 Brix. The material was then exposed to at least 2 mass equivalents of Dowex Monosphere 88 ion exchange resin. Exposure can occur by shaking a flask at 120-170 rpm or by filtration through a wet slurry packed column with sufficient cooling time. residence so that the solution reaches a final pH between 3 and 5. The oligomer solution was isolated by filtration (as in the case of stirred reactions) or elution (as in the case of column filtration) and the process was repeated with Dowex Monosphere 77 ion exchange resin in an analogous manner until the pH of the solution was above 5.5. Finally, the solution was exposed to Dowex Optipore SD-2 Adsorbent decolorizing resin until the solution was sufficiently clarified and filtered through a 0.2 micrometer sterile filter to remove residual solids and possible microbiological contaminants. Glycans isolated from cellular matter reactions can be further purified by centrifugation, gel chromatography, ultrafiltration or dialysis, precipitation or other well-known methods to remove residual cellular matter.

Example 17. Fractionation of glycan polymer preparations by activated carbon chromatography

If desired, any glycan generated as described herein is fractionated by activated carbon chromatography. Oligo- and polysaccharides were synthesized and fractionated into differential molecular weight groups by activated carbon chromatography. 500 mg of a 40-60 Brix aqueous glycan solution and 6 g of activated carbon (glycan:carbon = 1:12, w/w) were stirred in 100 ml of a 1% v/v ethanol/water solution. . The mixture was stirred at 300 rpm in a 250 mL beaker at room temperature for 3 h and vacuum filtered through a thick glass frit preloaded with 6 g of celite 545 (Acros Organics). The charcoal and celite were mixed finely and loaded into an empty 20 mL Biotage Samplet cartridge. The outlet of the cartridge was connected to the inlet of a second 20 mL Biotage Samplet cartridge prefilled with 8 g of charcoal/celite mixture (1:1, w/w). Then, 100 ml of increasing concentrations of ethanol/water eluents (0%, 1%, 3%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%) were passed through. , 40%, 45%, 50%, v/v) through the cartridge column at a flow rate of 3.7 ml/min using a Masterflex peristaltic pump to desorb the glycan fractions. 100 ml of eluate was collected for each fraction and dried under vacuum.

In one embodiment, this process was used to remove only monomeric sugars from the glycan. In this embodiment, the carbon was washed with 1% aqueous ethanol until the monomer no longer eluted from the column. The charcoal was then washed with 50% aqueous ethanol to remove all residual glycan as shown in Figure 11 .

In a second embodiment, this process was used to divide the glycan into multiple groups of increasing average molecular weight. In this embodiment, the carbon was washed sequentially with eluents of increasing proportion of ethanol (e.g., 1%, 2%, 5%, 10%, 20%, 50%). Fractions of increasing molecular weight were collected as the fraction of ethanol in the eluent increased.

Example 18. Removal of Monomeric and Dimeric Materials by Yeast Fermentation If desired, any glycan generated as described herein is removed of its monomeric and dimeric materials by yeast fermentation. Oligo- and polysaccharides were synthesized and purified to remove monomeric species and some dimeric species by fermentation with a yeast culture. This procedure is primarily intended to remove materials that may be digestible by the host's metabolism to enrich the abundance of glycans that are exclusively available for bacterial fermentation. Bread yeast (2 g, Fleischmann's yeast, Fenton, MO) was swollen in 10 mL deionized water for 1–16 h at 4 °C. Sodium alginate (2.5 g) was dissolved in 100 mL of hot deionized water by stirring at −300 rpm for 1 h on a hot stirring plate maintained at −80 °C. The final volume of sodium alginate solution was adjusted to 100 ml with deionized water. After the temperature of the alginate solution cooled to −30 °C, the yeast suspension was mixed with the alginate solution at −100 rpm for 10 minutes. The mixture was transferred to a 15 mL syringe fitted with a 21 gauge needle and slowly added dropwise to a 4% calcium chloride solution (Sigma) without stirring in a 200 mL Erlenmeyer flask to form beads. alginate with immobilized yeast. Then, the beads were kept at 4 °C for 1 h to solidify and washed at least 5 times with water to remove calcium chloride. This stock of yeast beads was kept at 4 °C in water for no more than 1 week. Yeast beads were added to 20 Brix glycan solutions at 37 °C for 24 h with a ratio of −6 ml of beads to 25 ml of glycan solution. The spent beads were replaced with new beads and incubated for another 39 h. The progression of monosaccharide elimination was monitored by SEC-HPLC. A second replenishment of beads was applied for samples requiring additional monosaccharide removal. After yeast treatment, the glycan solutions were filtered through a sterile 0.2 μm filter and passed through ion exchange columns as described in Example 16. The filtrates were collected and concentrated in vacuo. All monomeric galactose and glucose were removed from the glycans using this method. Some dimeric materials (e.g., maltose, lactose) could be removed using this method, although some dimers were resistant to fermentation (e.g., melibiose, allolactose). Figures 7A-7B show a maltose-derived glycan before and after yeast fermentation.

Bread yeast (Fleischmann's yeast, Fenton, MO) was selected to purify glycans prepared from maltose, sucrose, palatinose, cellobiose, melecitose, and melibiose. Kluyveromyces marxianus (ATCC 46537) was selected to purify glycans prepared from raffinose, gentiobiose, lactose and lactulose. Kluyveromyces marxianus beads were prepared analogously, except that the yeast suspension was prepared by mixing 10 g of freshly cultured yeast with 2.5 ml of water.

Example 19. Single Strain Growth Assays to Evaluate the Effects of Low DP Glycans An in vitro assay was performed to evaluate the ability of various bacterial strains, including commensals and gastrointestinal tract pathogens, to utilize enzymatically synthesized glycans (i.e., glycan polymers). ) and the sugars from which they were derived as growth substrates. The assay was designed to determine whether enzymatically synthesized glycans differ from the sugars from which they are derived in how well they support the growth of potentially beneficial or harmful bacterial members of the human microbiome. Bacterial strains were manipulated at all stages in an anaerobic chamber (AS-580, Anaerobe Systems) with a palladium catalyst. Initially, the chamber was made anaerobic by purging it with an anaerobic gas mixture of 5% hydrogen, 5% carbon dioxide and 90% nitrogen and was subsequently maintained in an anaerobic state using this same anaerobic gas mixture. Chamber anaerobicity was confirmed daily using Oxoid anaerobic indicator strips that change color in the presence of oxygen. All culture media, assay plates, other reagents and plastic consumables were pre-reduced in the anaerobic chamber for at least 24-48 hours before contact with bacteria. The enzymatically synthesized lacto-oligo, melib-oligo, malto-oligo, sucro-oligo, raffinooligo and lactulo-oligo glycans and the sugars from which they were derived, lactose, melibiose, maltose, sucrose, raffinose and lactulose, were sterilized by filtration. were prepared at 5% w/v in sterile water and added to Costar 3370 assay plates for a final concentration of 0.5% w/v in the assay, with each glycan analyzed in three non-adjacent wells and dextrose and water supplied as positive and negative controls.

Bacterial isolates were obtained from the American Type Culture Collection (ATCC) and the Leibniz Institute DSMZ-German Institute for Microorganisms and Cell Cultures (DSMZ). Cultures of the Bacteroidetes Bacteroides thetaiotaomicronATCC 29741 “BTH.8” and Parabacteroides distasonisATCC 8503 “PDI.6” were grown; the clostridialesClostridium scindensATCC 35704 “CSC.32”,Dorea formicigeneransATCC 27755 “DFO.36”,dorea longicatenaDSM 13814 “DLO.76” andBlautia hanseniiATCC 27752 “BHA.20” anaerobically in minced meat glucose broth (CMG, Anaerobe Systems ), a pre-reduced enriched medium including lean ground beef, enzymatic digest of casein, yeast extract, potassium phosphate, dextrose, cysteine, hemin and vitamin K1, for 18-48 hours at 37°C. Inocula were prepared by determining the optical density of each culture at 600 nM (OD600) in a Costar 3370 polystyrene 96-well flat-bottom assay plate using a Biotek Synergy 2 plate reader with Gen52 all-in-one microplate reader software. 0 according to the manufacturer's protocol and diluting the cells to final OD6000.01 in defined and semi-defined media that were prepared without sugars. Isolates ofD. formicigenerans, P. distasonis, B. hanseniiyD. longicatenaen sodium chloride 900 ml/l, calcium chloride dihydrate 26 ml/l, magnesium chloride hexahydrate 20 ml/l, manganese chloride tetrahydrate 10 ml/l, ammonium sulfate 40 ml/l, iron sulfate heptahydrate 4 ml /l, cobalt chloride hexahydrate 1 ml/l, potassium phosphate dibasic 300 ml/l, sodium phosphate dibasic 1.5 g/l, sodium bicarbonate 5 g/l, biotin 0.125 ml/l, pyridoxine 1 ml/l l, pantothenate 1 m/l, histidine 75 ml/l, glycine 75 ml/l, tryptophan 75 ml/l, arginine 150 ml/l, methionine 150 ml/l, threonine 150 ml/l, valine 225 ml/l, isoleucine 225 ml/l, leucine 300 mg/l, cysteine 400 ml/l and proline 450 ml/l (Theriot CMet al.Nat Commun. 2014; 5:3114), supplemented with 0-10% (v/v) of CMG. It was testedB. thetaiotaomicronen buffer 100 mM potassium phosphate (pH 7.2), 15 mM sodium chloride, 8.5 mM ammonium sulfate, 4 mM L-cysteine, 1.9 μM hematin, 200 μM L-histidine, 1.9 μM hematin magnesium 100 |iM, iron sulfate heptahydrate 1.4 |iM, calcium chloride 50 |iM, vitamin K31 |ig/mk and vitamin B125 ng/ml (Martens ECet al.Cell Host & Microbe 2008; 4, 447-457 ). It was testedC. scindensen tryptone peptone 10 g/l, yeast extract 5 g/l, L-cysteine hydrochloride 0.5 g/l, potassium phosphate buffer 0.1 M, pH 7.2, vitamin K3 1 |ig/ml, 0.08% w/v calcium chloride, 0.4 |ig/ml iron sulfate heptahydrate, 1 |ig/ml resazurin, 1.2 |ig/ml hematin, 0.2 mM histidine, 0 Tween 80 .05%, 0.5% meat extract (Sigma), 1% trace element supplement (ATCC), 1% vitamin supplement (ATCC), 0.017% v/v acetic acid, 0.001% v/v isovaleric acid /v, 0.2% v/v propionic acid and 0.2% v/v N-butyric acid (Romano KAet al.mBio 2015; 6(2): e02481-14). Bacteria were exposed to the enzymatically synthesized lacto-oligo, melib-oligo, malto-oligo, sucrooligo, raffino-oligo and lactulo-oligo glycans and the sugars lactose, melibiose, maltose, sucrose, raffinose, lactulose and dextrose at a final concentration of 0.5% w/v in 96-well microplates, 200 |il final volume per well, at 37 °C for 18-48 hours, anaerobically. OD600 measurements were obtained for each isolate at the end of the incubation period using a Biotek Synergy2 reader with Gen52.0 software according to the manufacturer's specifications. Measurements for each strain were blanked by subtracting the average OD600 value of the strain in the absence of glycan or sugar. Table 8 and the following three paragraphs summarize the results obtained.

Table 8. Growth of intestinal commensals on enzymatically synthesized sugars and glycans.

Some enzymatically synthesized glycans support different levels of growth of intestinal commensal bacteria in the assay than the sugars from which they are derived. As seen in Table 8, raffino-oligo supported greater growth than raffinose in FirmicutesD. formicigenerans, C. scindens, D. longicatenatB. hansenii and BacteroideteB. thetaiotaomicronin the test. Lactulo-oligo supported greater growth than lactulose of D. formicigenerans, C. scindens, P. distasonis, B. thetaiotaomicronyD. longcatena in the test, while B. hansenii grew less well on lactulo-oligo than on lactulose in the trial. In the trial, sucro-oligo supported increased growth of D.formicigenerans, P. distasonis and B. hanseniique sucrose and lower growth ofB. thetaiotaomicronyD. longicatenathan sucrose. Enzymatically synthesized glycans also differ in the amount of growth they support among the bacterial strains in the assay. In the trial, raffino-oligo supported high levels of growth of P. distasonis, B. thetaiotaomicron and B. hanseniiwith OD600 values greater than 0.7, moderate growth levels ofD. formicigeneranswith OD600 values between 0.3 and 0.7, and a lower level of growth of C.scindensyD. long chain with OD600 values between 0.1 and 0.3.

In the assay, some enzymatically synthesized glycans also support different levels of enteric pathogen growth than the sugars from which they are derived. Sucro-oligo supported less than half of the growth of S.enterica, E. faecium and C. difficile than sucrose in the assay, while raffino-oligo supported greater growth of these pathogens than raffinose. Lactulo-oligo and lactulose supported similar growth levels of these pathogens in the assay.

Some enzymatically synthesized glycans support levels of bacterial growth in the assay similar to the sugars from which they are derived. In the trial, the bacterial commensalsD. formicgenerans, D.

longicatena, B. hansenii, C. scindens, P. distasonisyB. thetaiotaomicron grew to similar levels, with optical densities differing by 20% or less, in lacto-oligo and lactose, melib-oligo and melibiosa, and maltooligo and maltose. The enteric pathogens S.enterica, E. faecium and C. difficile also grew to approximately similar levels in the melib-oligo and melibiose, malto-oligo and maltose, and lacto-oligo and lactose assay, with the exception of C. difficil on lacto-oligo and lactose, for which there was a difference in growth. of 30%.

The differences observed in the assay between the activity of enzymatically synthesized glycans and the sugars from which they are derived with respect to the bacterial strains they support and the level of growth they support for various bacterial strains suggest that an enzymatically synthesized glycan can be administered to differentially support one or more beneficial microbes in vivo for therapeutic benefit. For example,C. scindensse has been associated with protection against BC-associated diarrhea. difficile (Buffieet al, Nature 2015). Administration of an enzymatically synthesized glycan that selectively promotes the growth of C. scindens can stimulate the growth of C. scindens in vivo and, therefore, be beneficial in the prophylaxis or treatment of diarrhea associated with C. difficile.

Example 20. Glycosidase profiles and sugar utilization of selected metabolite producers

Linking metabolic functions encoded by gut bacteria with carbohydrate utilization genes has the potential to screen in silico glycan polymer preparations to detect human diseases linked to specific metabolites. However, many metabolic functions have been shown to be polyphyletic and, therefore, 16S rRNA gene sequencing is not a suitable analysis method (Vitalet al.,2014, Martinez del Campo et al.,2015). In order to overcome this challenge, genomic and metagenomic analyzes can be used to directly analyze genes involved in metabolite synthesis and link these genes to carbohydrate utilization through sequenced genomes.

The Carbohydrate-Active enZYmes (CAZy; http://www.cazy.org/) database describes enzyme families with catalytic or carbohydrate-binding modules for enzymes that degrade, modify, or create glycosidic bonds. Glycoside hydrolases (GH) and glycosyltransferases (GT) are important enzymes for carbohydrate utilization in the gut microbiome. CAZy families are widely distributed phylogenetically (Kaoutariet al., 2013).

In this analysis, a total of 439 sequenced genomes isolated from the gastrointestinal tract were used as part of the Human Microbiome Project (http://hmpdacc.org/reference_genomes/reference_genomes.php). All genomes were annotated using the Carbohydrate-Active enZYmes (CAZy; http://www.cazy.org/) database using USEARCH v8 and the -usearch_global option, requiring 90% global identity to the target protein. . Additionally, all genomes were annotated for their predicted involvement in 1) butyrate production, 2) conversion of choline to TMA via TMA-lyase, and 3) conversion of urea to ammonia. For butyrate production, annotation focused on the acetyl-CoA pathway, the most abundant pathway encoded in the gut microbiome (Vitalet al., 2014). In order to predict butyrate production, the terminal genes in the (butybuk) pathway were used, requiring an overall 90% identity with a target protein identified in the genome analysis by Vitalet al. In order to predict TMA-lyase potential, BLASTP was performed on the gencutCgen described in Martínez del Campo et al., which requires >60% identity in more than 40% of the target and query proteins. After annotation of both metabolite production and CAZy content, enrichment of carbohydrate utilization genes into metabolite producers (butyrate) or non-metabolite producers (TMA) was performed using the Wilcox test with correction by FDR. For conversion of urease, indole, p-cresol, propionate, and secondary bile acids, all HMP genomes were annotated using the KEGG database, with more than 70% global identity using the -usearch_global option. For urease production, EC number 3.5.1.5 was used as a urease enzyme marker to identify urease-positive genomes. For the production of indole. EC number 4.1.99.1 was used as a marker for the tryptophanase enzyme to identify indole-positive genomes. For the production of p-cresol, EC numbers 4.1.1.83, 2.6.1.-, 4.1.1.-, 1.2.7.5 were used to identify the route from tyrosine to p-cresol via 4-phenylhydroxyacetate. For propionate, EC numbers 6.4.1.3, 2.1.3.1, 4.1.1.41, 1.2.1.27, 2.3.3.5, 1.2.1.87, 1.3.1.95, 1.3.8.7, 2.3.1.54, 2.3.1.168, 2.3 were used .1.8, 2.3.1.222 to identify all propionate production routes. For secondary bile acid conversion, the following EC numbers were used to identify bile acid converters: 1.1.1.159, 1.1.1.176, 1.1.1.201, .1.1.238, 1.1.1.391, 1.1.392, 1.1.393 , 1.1. 395, 1.1.1.52, 2.8.3.25, 4.2.1.106, 6.2.1.7.

A total of 439 genomes sequenced across a variety of commensal bacterial species isolated from healthy human gut microbiomes as part of the Human Microbiome Project were predicted to have the capacity to produce butyrate, convert urea to ammonia via urease, and convert choline to TMA (Figure fifteen). Each of these three important bacterial functions is widely but discontinuously distributed in the phylogeny, demonstrating the importance of a functional approach to characterizing the metabolic potential of a microbiome. For example, although butyrate production is predicted to be universal in species of Faecalibacterium and Roseburia, species of Eubacteria and Lacnospiraceae appear to be more variable in their ability to produce butyrate. Furthermore, the production of these three metabolic functions appears to be generally non-overlapping between species, presenting the potential for selective production of beneficial and simultaneous suppression of toxic metabolites.

A total of seven CAZy families and subfamilies were identified as differentially abundant between butyrate producers and predicted non-producers (Figures 16A-16B; P<0.001, Wilcox rank sum with FDR correction). Of these families, two are almost completely absent from the non-butyrate producers (GH13.36 and GH113), which are known to target glucose- and mannose-containing glycans, respectively (Figures 16A-16B; Table 9 below). In general, enzymes that are enriched in butyrate producers target glycans that comprise glucose, mannose, and galactose, with glucose being the most common target composition (Table 9). This finding is supported experimentally, using both a vivo fecalex community and an in vitro defined community cultured on various glycan compositions and assayed for SCFA production. To identify the monomer compositions and interactions that most contribute to increased SCFA production, including butyrate, a LASSO (glmnet in R) linear regression model was used with cross-validation minimum lambda for defined communities and maximum lambda within a minimum lambda standard error for ex-vivo) with percent monomer composition as input, allowing for all second-order interaction terms. All positive model coefficients are shown in Figures 22A-22B. In this case, glucose is an important predictor for the production of all SCFAs, including butyrate, for defined communities (Figure 22B), and the combination of glucose, galactose and mannose increases butyrate production in fecalex-vivo communities, while glucose alone is predicted to increase acetate production (Figure 22A). In addition to supporting the use of a target monomer composition as a predictor of metabolic potential, using a LASSO linear regression model with percent monomer composition as input to predict the growth of individual bacterial strains, it is shown that the monomer composition is highly predictive of bacterial growth. Although these findings support the glycan target genes that are most differential between butyrate producers and non-producers, they are not exhaustive of the most abundant glycosyl hydrolases encoded in butyrate producers (Figure 17).

Table 9. Target glycan composition for CAZy families enriched in butyrate producers relative to non-butyrate producers.

This method can further be applied to target glycans for metabolites that are harmful and desirable to decrease, including TMA and ammonia. For example, 11 CAZy families and subfamilies were identified that are exclusive and overrepresented in taxa that do not encode the genecutc (Figures 18A-18B; P<0.05, Wilcox rank sum, corrected by FDR), which have been demonstrated that it is responsible for converting choline into TMA in the intestine (Martínez del Campo, et al). Furthermore, at least one of these families is encoded in almost 50% of bacterial species that do not also encode gencutc. These families They target a broader range of glycan compositions (Figures 18A-18B, Table 10), however, galactose and glucose are the most abundant target monomer compositions. These CAZy families and subfamilies are the most differentially abundant between paracutC positive and negative genomes, however, another set of genes can be found to be the most abundant in paracutC negative genomes and are also desirable targets for TMA reduction (Figure 19).

Table 10. Target glycan composition for CAZy families in genomes lacking the TMA-lyase enzyme.

Extending this application to the production of ammonia cleavage from urea by the enzyme urease, 7 CAZy families and subfamilies were identified that are differentially encoded between urease positive and negative genomes (Figures 20A-20B; P<0.05, rank sum Wilcox, corrected by FDR).

Furthermore, these enzymes are unique to urease-negative genomes and are exclusive targets of glucose- and galactose-containing glycans (Figures 20A-20B, Table 11). These CAZy families and subfamilies are the most differentially abundant between urease-positive and -negative genomes; However, another set of genes can be found to be the most abundant in urease-negative genomes and are therefore also desirable targets for ammonia reduction (Figure 21).

Table 11. Target glycan composition for CAZy families in genomes lacking the urease enzyme.

Extending this application to propionate production, 7 CAZy families and subfamilies were identified that are differentially encoded between propionate producers and non-propionate producers (Figures 30G, P<0.01, Wilcox rank sum, FDR corrected). These enzymes are enriched in non-propionate producers and are targets of N-acetylglucosamine, xylose, arabinose, glucose, galactose and fucose (Figure 30G, Table 25). These CAZy families and subfamilies are the most differentially abundant between propionate producers and non-producers; However, another set of genes can be found to be the most abundant in non-propionate producers and are therefore also desirable targets for propionate reduction (Figure 30H).

Table 25. Target glycan composition for CAZy families in genomes lacking propionate production pathways.

Extending this application to bile acid conversion, 6 CAZy families and subfamilies were identified that are differentially encoded between secondary bile acid converters and non-secondary bile acid converters (Figure 30A, P<0.05, Wilcox rank sum, corrected by FDR). These enzymes are enriched in secondary bile acid converters and are exclusive targets of trehalose, sialic acid and glucose (Figure 30A, Table 26). These CAZy families and subfamilies are the most differentially abundant among secondary bile acid converters and non-secondary bile acid converters; However, another set of genes may be found to be the most abundant in non-propionate producers and are therefore also desirable targets for secondary bile acid conversion (Figure 30B).

Table 26. Target glycan composition for CAZy families in genomes with secondary bile acid synthesis pathways.

Extending this application to indole production, 7 CAZy families and subfamilies were identified that are exclusively encoded in non-indole-producing bacteria (Figure 30C). These enzymes are encoded exclusively in non-indole-producing bacteria and are enriched for glucose, xylose, arabinose, and mannose targets (Figure 30C, Table 27). These CAZy families and subfamilies are encoded exclusively in non-indole-producing bacteria; However, another set of genes can be found to be the most abundant in non-indole-producing bacteria and are therefore also desirable targets for indole reduction (Figure 30D).

Table 27. Target glycan composition for CAZy family in genomes lacking a tryptophanase encoded for indole production.

Extending this application to p-cresol production, 7 CAZy families and subfamilies were identified that are differentially encoded between p-cresol producers and non-p-cresol producers (Figure 30<e>, P<0.01, sum of Wilcox ranges, corrected by FDR). These enzymes are enriched in non-p-cresol-producing bacteria and enriched for xylose, arabinose, galactose and glucose targets (Figure 30E, Table 28). These CAZy families and subfamilies are encoded exclusively in non-indole-producing bacteria; However, another set of genes can be found to be the most abundant in non-p-cresol-producing bacteria and are therefore also desirable targets for p-cresol reduction (Figure 30F).

Table 28. Target glycan composition for CAZy families in genomes lacking an encoded pathway for p-cresol production.

For all strains that grow well on synthesized glycans, the monomer composition is strongly predictive of the growth of the strains on a particular glycan as described in Table 12.

Table 12. R2 calculated using a LASSO linear regression model on monomer composition.

Example 21. Targeted Enrichment of a Microorganism Using an Exemplary Glycan Polymer Preparation

If a microbial species of interest is present in a microbial community, you may observe increased representation of that species by contacting the microbial community with certain glycan polymer preparations. For example, it is known that species of the genus Bacteroides have the highest amount of carbohydrate-active enzymes (CAZymes). Among those species, the genome sequence of Bacteroides cellulolyticus comprises the largest number of genes encoding enzymes dedicated to carbohydrate degradation of all known sequenced genomes of the genus: 503 CAZyme in total including 373 GH, 28 carbohydrate esterases. carbon and 84 glycosyltransferases (McNultyet al, PLOS Biology, doi:10.1371/joumal.pbio. 1001637). To show that the representation of Bacteroides cellulolyticus could be selectively increased in response to administration of an exemplary glycan polymer preparation, a complex bacterial community comprising 15 bacterial strains was assembled and tested.

The composition of this defined community was: 1) Phylum Actinobacteria: 2 strains of Bifidobacterium longum; 2) Phylum Firmicutes: Blautia hansenii, Clostridium nexile, Clostridium scindens, Dorea formicigenerans, Dorea longicatena, Ruminococcus obeum; 3) Phylum Bacteroidetes: Bacteroides caccae, Bacteroides cellulolyticus, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Prevotella copri, Para bacteroides distasonis; 4) Phylum Verrucomicrobia: Akkermansia muciniphila.

Each strain was grown separately in conventional minced meat glucose medium for 18 hours. Each culture was diluted to a final optical density (OD600) of 0.01 and combined into the final mixed community. This community was cultured for 48 hours in a medium supplied with the following carbohydrates as the sole carbohydrate source: glucose, FOS, Glu100 (also called “Glu”), Glu50Gal50 (also called “GluGal”), Gal100 (also called called “Gal”) and Man52Glu29Gal19 (also called “ManGluGal”). Water was added to a medium without any added carbon source as a control.

The resulting community, as well as individual strains, were added to a 96-well plate (Costar flat bottom, 300 μl) containing growth medium without glycan, or with each of the glycans listed above. The final concentration of each glycan in the assay was 5%. Each glycan was represented 3 times within each growth plate and was the only carbon source for the bacteria. Plates were incubated at 37°C in an AS-580 anaerobic chamber for a total of 48 hours.

At 18 and 48 hours, the optical density was determined for each community incubated with a glycan, and they were divided into 150 ul culture aliquots and immediately frozen at -80 °C for 16S rRNA gene sequencing to determine the changes in community composition as a function of time and glycan.

To determine the representation of each strain in this defined community, genomic DNA was extracted from each community and the 16S rRNA gene was amplified and sequenced. As shown in Figures 23A-23B, the relative abundance of Bacteroides cellulolyticus increased from an average of 4% to 30% when a defined community composed of 15 strains was grown on Glu100 glycan, but not on other carbon sources.

Furthermore, when a community was grown in the presence of a carbon source other than Glu100, as shown in Figure 23A, and Glu100 was then added to an established community, the relative abundance of Bacteroides cellulolyticus increased from an average of 4% to 14%, as shown in Figure 23B (Gal100, Glu50Gal50, Man52Glu29Gal19). Importantly, when B. cellulolyticusa a community during a stationary phase, such as communities grown in the presence of glucose or FOS, no changes were observed inB. cellulolyticus, implying that other growth-limiting nutrients, such as nitrogen or phosphorus sources, have probably been consumed (Figures 23A-23B). The data suggest the feasibility of targeted enrichment of specific taxa in a bacterial community using the glycan polymers described herein.

Example 22. Demonstration of a symbiotic administration to an established in vitro defined community

In certain diseases, the native microbiota can be altered by the ingestion of antibiotics or other agents that are harmful to the microbiota. Administration of probiotic species is a common attempt to restore the native microbiota. However, most FDA-approved probiotics are lactic acid bacteria that are present in very low abundance in the gut microbiota of adults. Furthermore, administration of these species does not result in the restoration of commensal microbiota, such as members of the genus Bacteroides. Furthermore, grafting a probiotic species into an established complex community is often difficult. Therefore, simultaneous administration of a probiotic along with a carbon source that selectively consumes that probiotic species may be necessary to ensure successful engraftment of this species into a resident community. Herein, successful engraftment of Bacteroides cellulolyticus is demonstrated in an established community, where the community was significantly enhanced by simultaneous administration of an exemplary glycan polymer preparation (Glycan Glu100).

A defined community composed of 14 strains (the 15 strains listed in Example 21 except Bacteroides cellulolyticus) was grown in the presence of 6 glycans or water as the sole carbon source for 18 hours. At the 18th hour, a culture of Bacteroides cellulolyticusa at OD=0.1 was added to each community. To test whether engraftment of Bacteroides cellulolyticus was dependent on its preferred carbon source (Glu100), 20 uL of 5% Glu100 or Bacteroides cellulolyticus and 20 uL of 5% Glu100 were added to each established community, as shown in Table 13. .

��� At 48 hours, the optical density was determined for each community, aliquots of 150 ul of culture were divided and immediately frozen at -80 °C for 16S rRNA gene sequencing to determine changes in composition of the community as a function of time and glycan.

To determine the representation of each strain in this defined community, genomic DNA was extracted from 150 μl of cultures using the MoBio Microbiome<a>D<n>/RNA extraction kit (catalog number 27500-4-EP). V4 region of the 16S rRNA gene was amplified using forward 515 and reverse 806 primers as described in Caporaso JGet al,ISME J. Amplicons were sequenced using the Illumina MiSeq instrument with 250 bp long reads using paired-end chemistry. Operational Taxonomic Units (OTUs) were collected using 97% sequence identity. Fifteen OTUs matching 16 strains were obtained. Two Bifidobacterium longum strains could not be resolved by the 16S rRNA gene, so 1 OTU represented both strains.

As shown in Figure 24A, the relative abundance of Bacteroides cellulolyticus was 8% in a community grown with Glu100 or water and only 2% for communities grown with other glycans.

However, when Glu100 was administered simultaneously with Bacteroides cellulolyticus, the relative abundance of this strain increased from 2% to 10% on average if a community had been growing in the presence of a glycan polymer preparation, or 25% if a community had been growing in the presence of a glycan polymer preparation. been growing in the absence of a glycan polymer preparation (Figure 24B). Importantly, if B. cellulolyticusa a community during a stationary phase, such as communities grown in the presence of glucose or FOS, no change was observed inB. cellulolyticus, implying that other growth-limiting nutrients, such as nitrogen or phosphorus sources, have probably been consumed (Figures 24A-24B).

Example 23: Ex vivo assay to evaluate the ability of glycan polymer preparations to modulate bacterial growth and metabolite production

An ex vivo assay was performed to evaluate the ability of a human fecal community, in an in vitro environment, to utilize different glycans. The ex vivo assay was designed to determine whether glycans can be used to differentially modulate a complex bacterial community and short-chain fatty acids, including those associated with protective effects. Fecal samples and suspensions were manipulated in an anaerobic chamber (AS-580, Anaerobe Systems) with a palladium catalyst. The glycans were prepared: , glu100, gal80man20, gal40ara60, gal80ara20, gal60man40, glu80gal20, xyl60ara40, glu80xyl20, glu60man40, glu20xyl80, glu20man80, man60ara40, glu80man20, glu60xyl40, gal20man80, gal60xyl40, gal80xyl20, glu40ara60, glu60ara40, xyl20ara80, man52glu29gal19, gal40xyl60, glu40xyl60, xyl100, glu20ara80, gal20ara80, gal20xy180, man20ara80, man100, xyl40ara60, ara100, xyl80ara20 and a control commercially available FOS (Nutraflora FOS; NOW Foods, Bloomingdale IL) at 5% w/v in water, were sterilized by filtration and added to 96 deep-well microplate assay plates for a final concentration of 0.5% w /v in the assay, with water supplied as positive and negative controls.

A human fecal sample donation was stored at −80°C. To prepare working stocks, the fecal sample was transferred to the anaerobic chamber and allowed to thaw. The fecal sample was prepared at 20% w/v in phosphate-buffered saline (PBS) pH 7.4 (P0261, Teknova Inc., Hollister, CA), 15% glycerol, and stored at −80°C. The 20% w/v 15% glycerol fecal suspension was centrifuged at 2,000 xg, the supernatant was removed and the pellet was suspended in sodium chloride 900 mg/l, calcium chloride dihydrate 26 mg/l, magnesium chloride hexahydrate 20 mg/l, manganese chloride tetrahydrate 10 ml/l, ammonium sulfate 40 ml/l, iron sulfate heptahydrate 4 ml/l, cobalt chloride hexahydrate 1 ml/l, potassium phosphate dibasic 300 ml/l, phosphate dibasic sodium 1.5 g/l, sodium bicarbonate 5 g/l, biotin 0.125 ml/l, pyridoxine 1 ml/l, pantothenate 1 m/l, histidine 75 ml/l, glycine 75 ml/l, tryptophan 75 ml/l, arginine 150 ml/l, methionine 150 mg/l, threonine 150 ml/l, valine 225 ml/l, isoleucine 225 ml/l, leucine 300 ml/l, cysteine 400 ml/l and proline 450 ml/l (Theriot CMet al.Nat Commun. 2014; 5:3114) to a 1% w/v fecal suspension. The prepared 1% w/v fecal suspension was exposed to the glycans glu33gal33man33, gal33man33ara33, glu50gal50, glu33gal33ara33, gal100, glu40man60, gal33man33xyl33, glu40gal60, glu20gal80, glu60gal40, glu80ara20, gal40man60, man 80xyl20, gal60ara40, glu100, gal80man20, gal40ara60, gal80ara20 x yl20ara80, man52glu29gal19, gal40xyl60, glu40xyl60, man60xyl40, xyl 100, glu20ara80, gal20ara80, gal20xy180, man20ara80, man100, xyl40ara60, ara100, xyl80ara20 and commercial FOS at a final concentration of 0.5% w/v in 96 deep-well microplates, 500 |il final volume per well, at 37 °C for 45 hours, so anaerobic. After incubation, the test samples were divided, one part was used for DNA extraction and sequencing and the other for short-chain fatty acid analysis.

Genomic DNA was extracted from glycan-treated fecal suspensions and controls, and variable region 4 of the 16S rRNA gene was amplified and sequenced (Earth Microbiome Project protocol www.earthmicrobiome.org/emp-standard-protocols/16s/ and Caporaso JGet al.Al. 2012. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms ISME J.). Operational Taxonomic Units (OTUs) were generated by aligning 16S rRNA sequences with 97% identity.

Glycan-treated and control fecal suspensions were centrifuged at 4000 x g for 10 min at 4°C and supernatants were transferred to new tubes. Samples were kept frozen at −80 °C until analysis. Samples were removed from the freezer and thawed. 1 ml of the culture supernatant was transferred to a glass vial. The pH of the suspension was adjusted to 2-3 by adding 100 μl of 50% sulfuric acid. The acidified samples were kept at room temperature and stirred for 10 minutes. For the extraction of volatile compounds, 50 μl of the internal standard (1% 2-methylpentanoic acid solution) and 1000 μl of anhydrous ethyl ether were added. The tubes were mixed end-to-end for 10 minutes and then centrifuged at 2000 g for 2 minutes. 1 μl of the top ether layer was injected into the chromatogram for analysis.

Chromatographic analysis of SCFA concentration was carried out using an Agilent 7890B system with a flame ionization detector (FID) (Agilent Technologies, Santa Clara, CA). A 30 m x 0.25 mm high-resolution gas chromatography capillary column coated with a film thickness of 0.25 |im (DB-FFAP) was used for the volatile acids (Agilent Technologies). Nitrogen was used as a carrier gas. The oven temperature was 145 °C and the FID and injection port were set to 225 °C. The volume of sample injected was 1 ul and the execution time for each analysis was 12 minutes. Chromatograms and data integration were carried out using OpenLab ChemStation software (Agilent Technologies). The results are summarized in tables 14 and 15.

The relative OTU abundances of glycan-treated fecal suspension samples were compared to the group without added glycans using the nonparametric Wilcoxon rank sum test to determine differences in microbiome compositions at the species level. In order to explore the associations between the species composition and the levels of butyrate, acetate and propionate in different glycans, first the proportion of species present in the group treated with glycans was normalized with respect to those obtained from the group (from control) without added carbon. The fold change values, thus obtained for each glycan treatment, were then correlated with the butyrate levels obtained from the glycans using the Spearman rank correlation method [Myles Hollander and Douglas A. Wolfe (1973), Nonparametric Statistical Methods. New York: John Wiley & Sons. Pages 185-194 (Kendall and Spearman Tests)]. Associations between species and butyrate levels were identified with statistical significance (Benjamini Hochberg corrected p value < 0.05) [Benjamini, Y. and Hochberg, Y. (1995) Journal of the Royal Statistical Society Series B 57, 289 300 ], and graphs representing the strongest associations are shown in Figures 25A-25D, 26A-26F and 27A-27D.

Table 14. Glycans differentially modulate linear short-chain fatty acid production in the ex vivo assay relative to the average glycan-free control.

As shown in Table 14, 48 synthetic glycans differentially modulate the production of the short chain unbranched fatty acids butyrate, acetate and propionate relative to the average no added glycan control in the assay. In the assay, the largest fold increases were observed with butyrate and propionate, as 1 glycan increased butyrate by at least 10-fold, 4 glycans increased propionate by at least 10-fold, and none of the glycans increased acetate by at least 10-fold. In the assay, 3-10-fold changes relative to the no-glycan control were observed in butyrate with 38 glycans, in acetate with 28 glycans, and in propionate with 27 glycans. In the assay, less than 3-fold changes relative to the no-glycan control were observed in butyrate with 9 glycans, in acetate with 20 glycans, and in propionate with 17 glycans.

Butyrate was modulated in the assay to varying degrees depending on the glycan composition. Butyrate increased at least 10-fold in the assay relative to the average no-added-glycan control with a trimer where 3 of the inputs were one-third each of mannose, galactose, and glucose. Butyrate increased at least 5-fold in the assay with glycans containing glucose and galactose, where glucose constituted less than 80% of the monomer input, and with glycans composed of three monomers, where two of the inputs were at least one-third of each of mannose and galactose. Butyrate increased less than five-fold in the assay with two out of four glycans composed of arabinose and xylose, and with four out of five glycans where the input was at least 80% arabinose.

Acetate was modulated in the assay to varying degrees depending on the glycan composition. Aside from FOS, glycans that increased acetate at least 5-fold in the assay included glucose as a monomer input. Apart from man100 and gal 100, glycans that increased acetate less than 3-fold in the assay included arabinose and/or xylose as monomer input. Therefore, the monomer composition seems to influence the production of acetate in the assay, with the contribution of glucose favoring the production of acetate, and the contributions of arabinose and xylose tending to relatively reduce the acetate.

Propionate was modulated in the assay to varying degrees depending on the glycan composition. Three out of four glycans that increased propionate at least 10-fold in the assay included both galactose and mannose as inputs, and two out of four included xylose and mannose as inputs. Propionate increased at least 5-fold in the assay with 14 of 16 glycans including mannose as a monomer input. In the assay, 16 of 18 glycans with which propionate changed less than 3-fold included xylose and/or arabinose as monomer input. Therefore, the monomer composition seems to influence the production of propionate in the assay, with the contribution of mannose favoring the production of propionate, and the contributions of arabinose and xylose tending to relatively reduce propionate.

a a . os g gray hairs mo uan erencamen e a pro ucc n e c os fatty e ca ena cor a branched in the ex vivo assay relative to the average glycan-free control

As seen in Table 15, glycans differentially modulate the short chain branched fatty acids isovalerate, isobutyrate and isocaproate in the assay relative to the average no added glycan control. In the assay, at least 2-fold increases were observed in 5-glycan isobutyrate, 1-glycan isovalerate, and 3-glycan isocaproate. In the assay, less than 2-fold changes were observed in 36-glycan isobutyrate, 38-glycan isovalerate, and 25-glycan isocaproate. Decreases of at least 2-fold were observed in the assay in 6-glycan isobutyrate, in 9-glycan isovalerate, and in 4-glycan isocaproate. Consistent with levels below the limit of detection, no isobutyrate was detected in the assay with 2 glycans, no isovalerate was detected with 1 glycan, and no isocaproate was detected with 16 glycans.

In the assay, the modulation of short chain branched fatty acids appears to depend on the glycan composition. 4 of 5 glycans for which isobutyrate increased at least 2-fold in the assay included mannose in combination with glucose and/or galactose. All 6 glycans with which isobutyrate decreased in the assay at least 2-fold included galactose. The ability of glycans to selectively increase or decrease branched short chain fatty acids suggests that they may be administered to selectively modulate short chain fatty acids in vivo for therapeutic benefit. Modulation by short-chain fatty acid glycans in the assay is associated with changes in several taxa. As indicated in Figures 25A-25D, 16S rRNA sequencing analysis indicates that bacteria identified as Clostridiaceae, Turicibacter, Roseburia and Bacteroides fragilis were positively correlated with butyrate in the assay. Many members of the Clostridiaceae and Roseburia taxa have been identified as butyrate producers based on reported genomic analysis and an increase in their relative abundance may be directly related to an increase in butyrate in the assay. The correlation of TuricibacteryB. fragiliscon butyrate production may be related to indirect effects. Turicibacter has been reported to produce lactate (Bosshard p Pet al,DSEM 2002), and lactate is a precursor for butyrate synthesis (Bourriaud Cet al,JAM 2005; Belenguer Aet al,AEM 2007).B. fragilis produces acetate (Macfarlane Set al, PNS 2003), which has been shown to be used for butyrate production by bacteria including Roseburia (Duncan SHet al, BJN 2004). TuricibacteryB. fragilis may indirectly contribute to an increase in butyrate production by providing precursors that are transformed into butyrate by bacteria such as Clostridiaceae and Roseburia.

Modulation by acetate glycan in the assay was negatively correlated with some bacterial taxa and positively correlated with others. As indicated in Figures 26A-26F, taxa including Ruminococcaceae, Rickenellaceae, and Oscillospira were strongly negatively associated with acetate production in the assay. Taxa that were positively associated with acetate in the assay included Bacteroides uniformis, Clostridiaceae and Bacteroides ovatus. As indicated in Figures 27A-27D,B. ovatusyB. uniformis were also positively associated with propionate in the assay, while taxa that were most strongly negatively associated with propionate differed from those most strongly associated with acetate and included Bifidobacterium, Ruminococcus bromii, and Roseburia faecis. Thus, differential modulation of relative abundances of bacterial taxa by glycans in the assay appears to be associated with differential modulation of short-chain fatty acids.

Example 24: Ability of Glycan Polymer Preparations to Modulate Bacterial Growth of Spore-Forming Bacterial Taxa

An in vitro assay was performed to evaluate the ability of various spore-forming bacterial commensals from the gastrointestinal tract to use different glycans as growth substrates. This assay was designed to evaluate the ability of selected glycans to promote the growth of spore-forming intestinal commensals, which have been associated with a range of beneficial health effects. Bacterial strains were manipulated at all stages in an anaerobic chamber (AS-580, Anaerobe Systems) with a palladium catalyst. Initially, the chamber was made anaerobic by purging it with an anaerobic gas mixture of 5% hydrogen, 5% carbon dioxide and 90% nitrogen and was subsequently maintained in an anaerobic state using this same anaerobic gas mixture. The anaerobicity of the chamber was confirmed daily using Oxoid anaerobic indicator strips that change color in the presence of oxygen. All culture media, assay plates, other reagents and plastic consumables were pre-reduced in the anaerobic chamber for 24 to 48 hours before contact with bacteria. The glycans gal 100, glu20gal80, glu40gal60, glu50gal50, glu60gal40, glu80gal20, glu100, glu80man20, glu60man40, glu40man60, glu20man80, man100, xyl100, xyl80ara20, xyl60ara40, 0, xyl20ara80, ara100, glu80ara20, glu60ara40, glu40ara60, glu20ara80, glu80xyl20 , glu60xyl40, glu40xyl60, glu20xyl80, gal80man20, gal60man40, gal40man60, gal20man80, gal80xyl20, gal60xyl40, gal40xyl60, gal20xy180, gal80ara20, gal60ara40, gal40ara60, gal20ara80, 0, man60xyl40, man40xyl60, man20xyl80, man80ara20, man60ara40, man40ara60, man20ara80, glu33gal33ara33, man52glu29gal19 , glu33gal33man33, glu33gal33xyl33, gal33man33xyl33, gal33man33ara33 and a commercially available control, FOS (Nutraflora FOS; NOW Foods, Bloomingdale IL) at 5% w/v in water, were filter sterilized and added to Costar 3370 assay plates for a concentration final 0.5% w/v in the assay, testing each glycan in two non-adjacent wells and providing dextrose and water as positive and negative controls.

Bacterial isolates were obtained from the American Type Culture Collection (ATCC) and the Leibniz Institute DSMZ-German Institute for Microorganisms and Cell Cultures (DSMZ). Cultures of Clostridium scindens ATCC 35704 “CSC.32”, Clostridium nexile ATCC 27757 “CNE.31”, Blautia producta ATCC 27340 “BPR.22”, Dorea longicatena DSM 13814 “DLO.76”, Ruminococcus obeum ATCC 29714 “ROB.74” and Blautia hansen were grown. iiATCC 27752 “BHA.20” anaerobically in ground beef glucose broth (CMG, Anaerobe Systems), a pre-reduced enriched medium that includes lean ground beef, casein enzymatic digest, yeast extract, potassium phosphate, dextrose, cysteine, hemin and vitamin K1, for 18-48 hours at 37 °C. Inocula were prepared by determining the optical density of each culture at 600 nM (OD600) in a Costar 3370 polystyrene 96-well flat-bottom assay plate using a Biotek Synergy 2 plate reader with Gen52 all-in-one microplate reader software. 0 according to the manufacturer's protocol and diluting the cells to final OD600 0.01 in defined and semi-defined media that were prepared without sugars. Isolates of B. hansenii, B. productayD. longicatenaen sodium chloride 900 ml/l, calcium chloride dihydrate 26 ml/l, magnesium chloride hexahydrate 20 ml/l, manganese chloride tetrahydrate 10 ml/l, ammonium sulfate 40 ml/l, iron sulfate heptahydrate 4 ml /l, cobalt chloride hexahydrate 1 ml/l, potassium phosphate dibasic 300 ml/l, sodium phosphate dibasic 1.5 g/l, sodium bicarbonate 5 g/l, biotin 0.125 ml/l, pyridoxine 1 ml/l l, pantothenate 1 m/l, histidine 75 ml/l, glycine 75 ml/l, tryptophan 75 ml/l, arginine 150 ml/l, methionine 150 ml/l, threonine 150 ml/l, valine 225 ml/l, isoleucine 225 ml/l, leucine 300 mg/l, cysteine 400 ml/l and proline 450 ml/l (Theriot CMet al.Nat Commun. 2014; 5:3114), supplemented with 0-10% (v/v) of CMG. C.scindens, C. nexileyR. obeumen tryptone peptone 10 g/l, yeast extract 5 g/l, L-cysteine hydrochloride 0.5 g/l, potassium phosphate buffer 0.1 M, pH 7.2, vitamin K31 |ig/ml, chloride calcium 0.08% w/v, iron sulfate heptahydrate 0.4 |ig/ml, resazurin 1 |ig/ml, hematin 1.2 |ig/ml, histidine 0.2 mM, Tween 80 at 0, 05%, 0.5% meat extract (Sigma), 1% trace element supplement (ATCC), 1% vitamin supplement (ATCC), 0.017% v/v acetic acid, 0.001% v/v isovaleric acid v, 0.2% v/v propionic acid and 0.2% v/v N-butyric acid (Romano KAet al.mBio 2015; 6(2): e02481-14). The bacteria were exposed to the glycans gal100, glu20gal80, glu40gal60, glu50gal50, glu60gal40, glu80gal20, glu100, glu80man20, glu60man40, glu40man60, glu20man80, man100, xyl100, xyl80ara20, xyl60ara40, ara60, xyl20ara80, ara100, glu80ara20, glu60ara40, glu40ara60, glu20ara80 , glu80xyl20, glu60xyl40, glu40xyl60, glu20xyl80, gal80man20, gal60man40, gal40man60, gal20man80, gal80xyl20, gal60xyl40, gal40xyl60, gal20xy180, gal80ara20, gal60ara40, gal40ara60, 80, man80xyl20, man60xyl40, man40xyl60, man20xyl80, man80ara20, man60ara40, man40ara60, man20ara80, glu33gal33ara33 , man52glu29gal19, glu33gal33man33, glu33gal33xyl33, gal33man33xyl33, gal33man33ara33, commercial FOS and dextrose at a final concentration of 0.5% w/v in 96-well microplates, 200 |il final volume per well, at 37 °C for 18-48 hours, anaerobically. OD600 measurements were obtained for each isolate at the end of the incubation period using a Biotek Synergy2 reader with Gen52.0 software according to the manufacturer's specifications.

Table 16. Glycan-supported growth of commensal spore formers.

As seen in Table 16, glycans support the growth of 6 spore-forming intestinal commensals in the assay to varying degrees. In the assay, gal100, glu20gal80, xyl80ara20, xyl60ara40, glu20xyl80 support the growth of the 6 strains; glu40gal60, glu50gal50, glu60gal40, xyl40ara60, xyl20ara80, glu40xyl60 and glu33gal33ara33 support the growth of 5 strains; xyl100, gal60ara40, man52glu29gal19 and glu33gal33man33 support the growth of 4 strains; and glu20ara80, gal20ara80, man40xyl60, man20xyl80, glu33gal33xyl33 and gal33man33ara33 support the growth of 3 strains.

Modulation of the growth of spore-forming commensals in the assay varies with glycan composition. Oligomers with inputs of glucose and galactose support at least half of the 6 strains in the assay, and the proportion of strains for which growth is supported in the assay generally increases as the proportion of galactose increases. In the assay, glu80gal20 and glu100 support 3 strains, glu50gal50 support 5 strains, and gal 100 and glu20gal80 support the growth of 6 strains. Among the 4 oligomers that include glu, gal, and another monomer as inputs, 3 oligomers (glu33gal33ara33, man52glu29gal19, and glu33gal33man33) support the growth of 4 strains in the assay, and 1 oligomer (glu33gal33xyl33) supports the growth of 3 strains in the assay. The number of strains supported in the assay by oligomers with contributions of xylose and arabinose and by oligomers with contributions of glucose and xylose increases as the proportion of xylose contribution increases. In the assay, glu80xyl20 supports 2 strains, glu60xyl40 supports 3 strains, glu40xyl60 supports 5 strains, and glu20xyl80 supports 6 strains. In the assay, oligomers containing contributions of xylose and arabinose support the growth of 5 or 6 strains, while xyl100 supports the growth of 4 strains and ara100 supports 2 strains.

The gastrointestinal tract of healthy humans contains a diverse microbial community that can include hundreds of different bacterial species. As described in US application 2016/0158294, the gut microbiota plays an important role in human health, with benefits including resistance to colonization against pathogens, regulation of host immune responses, production of essential nutrients and nutrient absorption, and maintenance of intestinal epithelial integrity. Under dysbiotic conditions, the gut microbiome population is altered and negative effects on the host may include altered immune responses, increased inflammation, altered metabolic responses, and increased susceptibility to pathogens. Probiotics represent only a few of the hundreds of bacterial species that are present in the normal, healthy human microbiota. Commensal spore-forming bacteria are abundant in the human microbiome and include species associated with various beneficial effects, including the production of butyrate, which is associated with the integrity of the intestinal epithelial barrier and the regulation of metabolic and immune responses, and protection against Clostridium difficile infection by C. scindens. Glycans can be administered to promote the growth of commensal spore-forming bacteria to provide a range of health benefits and prevent and treat diseases related to microbial dysbiosis.

Example 25: Glycosidase profiles and sugar utilization of spore-forming bacterial taxa

Glycoside hydrolases (GH) and glycosyltransferases (GT) were identified that are enriched in spore-forming bacterial taxa essentially as described in Example 20.

Genes from 40 glycoside hydrolase and transferase (CAZy) families and subfamilies were identified that are enriched in genomes of spore-forming bacteria compared to non-spore-forming bacteria (Figure 28, P < 0.05, Wilcox rank sum, corrected by FDR). The spore formers are summarized in table: 17.

��� For example, GH43, GH13 and GH28 were much more highly represented in genomes of spore-forming bacteria (Figure 28). Overenrichment also generally corresponded to a higher prevalence of detection in all genomes of spore-forming bacteria (Figure 29A). Overall, CAZyme families that were identified to be enriched in spore-forming bacteria were present in a larger portion of spore-forming bacterial genomes compared to non-spore-forming bacteria (Figure 29 B). These results show that specific carbohydrate-active enzymes are enriched in spore-forming bacteria. Table 18 shows the glycan monomers that are likely to be targeted by these enzyme families.

Table 18. Target glycan composition for CAZy families enriched in spore-forming bacteria relative to non-spore-forming bacteria.

Many target substrates containing glucose, xylose, galactose, and arabinose, suggesting that glycans with these constituents could be used to promote the growth of spore-forming bacteria. Glycans comprising glucose, xylose, galactose and/or arabinose promoted the growth of spore-forming bacteria in single-strain growth assays (see Example 24, Table 16).

Example 27: Enrichment of Specific Taxa Encoding Glycosidases in Human Fecal Suspensions in the Presence of Oligosaccharides Synthesized Using Target Glycosidases Glycan preparations were tested for their ability to modulate the levels of target bacterial species encoding specific glycosidases in a fecal suspension from a healthy human subject in vitro (also known as ex vivo assay). Samples and fecal suspensions were manipulated in an anaerobic chamber (AS-580, Anaerobe Systems) with a palladium catalyst. Glycans were prepared at 5% w/v in water, sterilized by filtration, and added to 96-well deep-well microplate assay plates for a final concentration of 0.5% w/v in the assay, with water supplied as positive and negative controls.

A human fecal sample donation was stored at −80°C. To prepare working stocks, the fecal sample was transferred to the anaerobic chamber and allowed to thaw. The fecal sample was prepared at 20% w/v in phosphate-buffered saline (PBS) pH 7.4 (P0261, Teknova Inc., Hollister, cA), 15% glycerol and stored at −80°C. The 20% w/v 15% glycerol fecal suspension was centrifuged at 2,000 xg, the supernatant was removed and the pellet was suspended in sodium chloride 900 ml/l, calcium chloride dihydrate 26 ml/l, magnesium chloride hexahydrate 20 ml/l, manganese chloride tetrahydrate 10 ml/l, ammonium sulfate 40 ml/l, iron sulfate heptahydrate 4 ml/l, cobalt chloride hexahydrate 1 ml/l, potassium phosphate dibasic 300 ml/l, phosphate dibasic sodium 1.5 g/l, sodium bicarbonate 5 g/l, biotin 0.125 ml/l, pyridoxine 1 ml/l, pantothenate 1 m/l, histidine 75 ml/l, glycine 75 ml/l, tryptophan 75 ml/l, arginine 150 ml/l, methionine 150 ml/l, threonine 150 ml/l, valine 225 ml/l, isoleucine 225 ml/l, leucine 300 mg/l, cysteine 400 ml/l and proline 450 ml/l (Theriot CMet al.Nat Commun. 2014; 5:3114) supplemented with 750 uM urea and 0.1% peptone in 1% w/v fecal suspension.

The prepared 1% w/v fecal suspension was exposed to melibiosa-1, melibiosa-enz19-1, melibiosa-enz20-1, melibiosa-enz16-1, melibiosa-enz17-1, raffinose-1, raffinose-enz19-1 , raffinose-enz16-1 at a final concentration of 0.5% w/v in 96-well deep-well microplates, 500 |il final volume per well, at 37°C for 14 hours, anaerobically. Melibiose and raffinose denote the substrate used to generate oligosaccharides with specific enzymes (enz19, enz20, etc.); and the number after the dash indicates a glycan preparation (e.g., -1) that has characteristics different from another glycan preparation (e.g., -3), which differ from each other within the ranges for the glycan preparations described in This document.

After incubation, genomic DNA was extracted from the glycan-treated fecal suspensions and controls, and the variable region 4 of the 16S rRNA gene was amplified and sequenced (Earth Microbiome Project protocol www.earthmicrobiome.org/emp-standard-protocols /16s/ and Caporaso JGet al.,2012. Ultrahigh-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms ISME J.). Raw sequences were demultiplexed and each sample was processed separately with UNOISE2 (Edgar 2016). Briefly, paired-end reads were merged and quality filtered. Unique reads were then denoised and the unfiltered merged sequences were mapped to denoised sequences. Taxonomy was assigned to the noise-free sequences using the RDP classifier (Wang 2007).

As demonstrated herein, when the glycosidases used to synthesize enzymes are encoded by gut microbial species, the resulting oligosaccharides enrich those specific species. For example, when glycosidases encoded by Lachnospiriaceae are used to generate oligosaccharides, the resulting oligosaccharides enrich Lachnospiriceae more than either the original substrate or oligosaccharides generated by enzymes encoded by different species (Figures 31A-31B). This is valid regardless of the substrate used to generate the oligosaccharide (Figures 31A-31B). This demonstrates a mechanism for targeting oligosaccharides to glycosidases in the gut microbiome to specifically enrich taxa and functions of interest.

They can target groups of unrelated taxa by identifying shared glycosyl hydrolases. As demonstrated herein, when using glycosyl hydrolases that are abundant in three unique genera, Bifidoabacteria, Bacteroides, and Roseburia, all 3 taxa are simultaneously enriched in the ex vivo community (Figures 32A-32D). This demonstrates the ability to target species from different phyla and additional target functions, or glycotaxons. The scope of the present embodiments described herein is not intended to be limited to the foregoing description, figures or examples, but rather to what is set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications can be made to this description without departing from the scope of the present invention, as defined in the following claims.

Claims (15)

1. Preparation of glycan polymer for use in a method of treating a subject having a disease or disorder associated with an unwanted level of a metabolite, wherein the metabolite is trimethylamine (TMA) or trimethylamine N-oxide ( TMAO), and wherein the disease or disorder is selected from the group consisting of atherosclerosis, cardiovascular disease, cardiovascular risk in HIV, carotid atherosclerosis, chronic heart disease, chronic heart failure, chronic kidney disease, chronic vascular disease, colorectal cancer, coronary heart disease, coronary artery disease (CAD), diabetes (type II), end-stage renal disease, HIV, inflammatory bowel disease, ischemic attack, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), obesity, induced acute bowel symptoms radiation (RIAIS) and stroke, understanding said method: select a glycan polymer preparation based on or with the knowledge that it modulates the production or level of the metabolite, and administering an amount of the glycan polymer preparation effective to result in a modulation of the level of the metabolite, thereby treating the disease or disorder, wherein the glycan polymer preparation comprises glucose and at least one alpha-glycosidic bond, optionally, wherein the alpha-glycosidic bond is alpha-1,3-glycosidic bond, alpha-1,4-glycosidic bond, or a combination thereof, and where the average degree of polymerization (DP) of the preparation is between DP3-15. 2. Preparation of glycan polymer for use according to claim 1, wherein the glycan polymer preparation comprises one, two, three, or all, of the following characteristics: Yo. The glycan polymer preparation further comprises glycan polymers comprising at least one beta-glycosidic bond, optionally wherein the beta-glycosidic bond is beta-1,3 glycosidic bond, beta-1,4 glycosidic bond or a combination thereof. themselves; ii. the glycan polymer preparation further comprises glycan polymers comprising galactose (e.g., a glu-gal preparation); iii. the glycan polymer preparation further comprises glycan polymers comprising mannose (e.g., a glu-man preparation); and iv. The glycan polymer preparation further comprises glycan polymers comprising galactose and mannose (for example, a glu-gal-man preparation). 3. Preparation of glycan polymer for use according to claims 1 or 2, wherein the glycan polymers, or at least 20, 30, 40, 50, 60, 70, 80, 90, 95 or 99% (in weight or number) of the glycan polymers, of the glycan polymer preparation, is a substrate for a glycosidase enzyme, wherein the glycosidase enzyme is optionally present in a human intestinal microbe, or wherein the human gut microbe is optionally a member of class 2 glycotaxons, bacterial taxa negative for the cutC gene. 4. Preparation of glycan polymer for use according to claim 3, wherein the glycan polymer is a substrate for a glycosidase enzyme selected from one or more of, for example, two, three, four, or more of a) family CAZy GT11, GT10, GH92, GH51, GH35, GH29, GH28, GH20, GH130, GH13 subfamily 8, or GH13 subfamily 14; either b) CAZy family GT2, GT4, GH2, GH23, GH3, GT8, GT51, GT9, GH1, GH92, GH73, GH31, GH20, GH28, GT25, GT28, GT35, GH18, GT0, GH13, GH36, GH97, GH105, GH25, GH4, GH32, GH78, GH29, GH0, GH51, GT10 or GH77. 5. Preparation of glycan polymer for use according to claim 1, wherein the disease or disorder is a chronic disease (for example, chronic kidney disease or end-stage renal disease, chronic heart disease, chronic heart failure, chronic vascular disease) , either wherein the disease or disorder is nonalcoholic fatty liver disease (NAFLD). 6. Preparation of glycan polymer for use according to claim 5, wherein the level of metabolite is decreased in the subject or a suitable sample of the subject having the disease or disorder, for example, decreased compared to a reference, e.g. For example, a predetermined baseline value, the level in the subject before treatment or a healthy control, optionally further comprising assessing the level of the metabolite, or a symptom of an unwanted level of the metabolite, for example, acquiring a level of the metabolite , optionally before treating the subject (for example, as a baseline), during treatment (for example, to monitor treatment success) and/or after treatment (for example, to assess relapse of the disease or disorder) . 7. Preparation of glycan polymer for use according to claim 6, wherein the level (e.g., systemic level, e.g. blood or fecal levels) of TMA is decreased (e.g., choline conversion rate or level). in TMA, for example, by gastrointestinal microbes, is reduced), for example, relative to a subject not treated with the glycan polymer preparation. 8. Preparation of glycan polymer for use according to any one of claims 1-7, further comprising selecting a subject for treatment based on or in response to acquiring knowledge of any one, two, three, four or more of: a) that the subject has an unwanted level of TMA or TMAO, b) that the subject has a disease or disorder (for example, a disease or disorder according to claim 5), c) that the subject has a gut microbiota dysbiosis (e.g., miscalibrated levels/relative abundance of, e.g. class 2 bacterial taxa according to claim 3), d) that the subject has responded to pretreatment with a glycan polymer (for example, a glycan polymer according to any of claims 1 or 2), e) that the subject has undergone therapy or other environment that results in dysbiosis, for example, antibiotic treatment, or gastric surgery prior to treatment, optionally comprising acquiring an appropriate value to determine the selection criteria. 9. Preparation of glycan polymer for use according to claim 8, wherein a suitable value can be acquired by analyzing a suitable biological sample from the subject, wherein the sample is optionally blood, feces, urine, saliva or an organic tissue sample. 10. Preparation of glycan polymer for use according to any one of claims 1-9, wherein the unwanted level of the metabolite is decreased (for example, in the subject or in a suitable sample taken from the treated subject) by 3%. , 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 50% after a treatment period (for example, compared to a reference, for example, a reference value predetermined, the level in the subject before treatment or a healthy control). 11. Preparation of glycan polymer for use according to any one of claims 1-10, wherein the treatment further comprises administering a second therapeutic agent (for example, a therapeutic agent other than the glycan polymer to treat the disease or disorder and /o to modulate the level of the metabolite), or administer a preparation of an intestinal microbe (for example, a human intestinal microbe), wherein the gut microbe (e.g., a human gut microbe) is preferably a class 2 (e.g., cutC gene-negative bacterial taxa), wherein the gut microbe is optionally selected based on its association with the metabolite (e.g. , based on its positive, negative correlation or lack of correlation with the metabolite), wherein the selection of the gut microbe optionally comprises choosing a gut microbe from Table 3 based on the association of the gut microbe with the metabolite (for example, based on its positive, negative correlation or lack of correlation with the metabolite). 12. Preparation of glycan polymer for use according to claim 11, wherein the glycan polymer is a substrate of the intestinal microbe (for example, a human intestinal microbe). 13. Preparation of glycan polymer for use according to any one of claims 1-12, wherein the glycan polymer is a substrate of an intestinal microbial glycosidase enzyme and promotes the growth of the intestinal microbe. 14. Preparation of glycan polymer for use according to any one of claims 1-13, wherein the glycan preparation a) is administered daily; b) is administered during a single treatment period, or c) is administered for more than one treatment period, for example, where one period between treatments is longer than one or both of the adjacent treatment periods or where one period between treatments is shorter than one or both of the adjacent treatment periods. 15. Glycan polymer preparation for use according to any of claims 1-14, wherein the glycan polymer is a substrate for a microbial constituent of the colon or intestine, wherein the glycan polymer preparation is optionally administered by orally or rectally.